HDAC Inhibition Upregulates PD-1 Ligands in …...Research Article HDAC Inhibition Upregulates PD-1...

Research Article

HDAC Inhibition Upregulates PD-1 Ligands inMelanoma and Augments Immunotherapy withPD-1 BlockadeDavid M.Woods, Andressa L. Sodr�e, Alejandro Villagra, Amod Sarnaik,Eduardo M. Sotomayor, and Jeffrey Weber

Abstract

Expression of PD-1 ligands by tumors and interaction withPD-1–expressing T cells in the tumor microenvironment canresult in tolerance. Therapies targeting this coinhibitory axis haveproven clinically successful in the treatment of metastatic mela-noma, non–small cell lung cancer, and other malignancies. Ther-apeutic agents targeting the epigenetic regulatory family of his-tone deacetylases (HDAC) have shown clinical success in thetreatment of some hematologic malignancies. Beyond directtumor cell cytotoxicity, HDAC inhibitors have also been shownto alter the immunogenicity and enhance antitumor immuneresponses. Here, we show that class I HDAC inhibitors upregu-lated the expression of PD-L1 and, to a lesser degree, PD-L2 inmelanomas. Evaluation of human and murine cell lines andpatient tumors treated with a variety of HDAC inhibitors in vitro

displayed upregulation of these ligands. This upregulation wasrobust and durable, with enhanced expression lasting past 96hours. These results were validated in vivo in a B16F10 syngeneicmurine model. Mechanistically, HDAC inhibitor treatmentresulted in rapid upregulation of histone acetylation of the PD-L1 gene leading to enhanced and durable gene expression. Theefficacy of combining HDAC inhibition with PD-1 blockade fortreatment of melanoma was also explored in a murine B16F10model. Mice receiving combination therapy had a slower tumorprogression and increased survival compared with control andsingle-agent treatments. These results highlight the ability ofepigenetic modifiers to augment immunotherapies, providing arationale for combining HDAC inhibitors with PD-1 blockade.Cancer Immunol Res; 3(12); 1375–85. �2015 AACR.

IntroductionMelanoma is associated with the highest mortality of any skin

cancer, and is generally fatal oncemetastatic disease develops (1).Overall response rates to decarbazine, the standard chemotherapyfor metastatic melanoma, are only 10%, with no demonstratedoverall survival (OS) benefit (2, 3). In recent years, the landscapeofmetastaticmelanoma treatment has been alteredby remarkableadvances in immunotherapeutic approaches. Targeting theimmune system by blocking checkpoint molecules has generateddurable responses, with overall responses rates of 10% to 15%and 30% to 40% when blocking CTLA-4 or PD-1, respectively(4, 5). These results led to the FDA approval of the anti–CTLA-4antibody ipilimumab in 2011 and the anti–PD-1 antibodiespembrolizumab and nivolumab in 2014.

Binding by PD-1 to the costimulatory ligands programmeddeath 1 ligand (PD-L1), also known as CD274 or B7-H1, and

programmed death 2 ligand (PD-L2), also known as CD273 orB7-DC, negatively regulates T-cell responses (6, 7). Mechanisti-cally, recruitment of phosphatases by ligation of PD-L1 to the PD-1 receptor on T cells reduces downstream phosphorylation, andthereby shuts off the function of molecules involved in thepathways that mediate TcR engagement, such as ZAP-70, PKC-q, andAkt (8, 9). As a result, proliferation and cytokineproductionare inhibited, ultimately leading to dysfunction or apoptosis of Tcells (8–11). Under normal conditions PD-L1 acts to temperimmune responses (12). Indeed, mice genetically deficient inPD-L1 signaling develop autoimmunity (13). However, in thecontext of cancer, expression of PD-L1 by tumors impairs tumor-reactive T cells and allows for immune escape. In melanoma, PD-L1 is upregulated in tumor and stromal cells, creating an immu-nosuppressive microenvironment (14). Similar to PD-L1, PD-L2is a critical negative regulator of T-cell responses (7, 15) and canbeupregulated on tumor cells and, more commonly, on antigen-presenting cells (7, 16).

Inhibition of the PD-L1–PD-1 pathway through the use of thePD-1–blocking antibodies pembrolizumab or nivolumab hasresulted in objective response rates in metastatic melanoma of21% to 45% (5, 17). The 2-year survival rate of treatment-refractory patients receiving nivolumab was 43% (18). In clinicaltrials targeting PD-L1, its blockade resulted in objective responserates of 17.3% in metastatic melanoma patients, with a 5.7%complete response rate (19). While showing some efficacy, it isclear that PD-L1 blockade has not achieved the same levels ofclinical benefit in melanoma as PD-1 blockade. Accumulatingevidence suggests that PD-L1 expression on tumor, but not on the

H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

D.M. Woods and A.L. Sodr�e share first authorship of this article.

Corresponding Authors: Jeffrey Weber, H. Lee Moffitt Cancer Center, 12902Magnolia Drive, Tampa, FL 33612. Phone: 813-745-2691. E-mail:[email protected]; and Eduardo M. Sotomayor,[email protected].

doi: 10.1158/2326-6066.CIR-15-0077-T

�2015 American Association for Cancer Research.

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surrounding immune infiltrate, is significantly associated withobjective response to PD-1–blocking antibodies (20). However,patient responses are observed in the absence of detectable PD-L1on tumors (21).

Histone deacetylases (HDAC) are key mediators of epige-netic regulation that act by removing acetyl groups from the N-acetyl lysine amino acid on the tail of histones, and haveproven to be attractive therapeutic targets for cancer. HDACsare phylogenetically classified as classes I, IIa, IIb, III, and IV.Class I HDACs, with the exception of HDAC8, participatein chromatin remodeling as components of multiprotein com-plexes (22). HDAC inhibitors represent a chemically diversegroup of drugs with several dozen HDAC inhibitors at variousstages of development. To date, the HDAC inhibitors vorinostatand romidepsin have been FDA approved for the treatmentof cutaneous T-cell lymphoma, and belinostat was recentlyapproved for the treatment of peripheral T-cell lymphomaand panobinostat for multiple myeloma. In addition, severalHDAC inhibitors are in mid-to-late-phase clinical trials, includ-ing studies of combination therapy for metastatic melanoma.

HDAC inhibitors induce cell-cycle arrest and apoptosis intransformed cells, including melanoma (23). In addition to theirselective cytotoxicity, HDAC inhibitors can induce awide range ofimmunologic changes in malignant cells. These changes includeincreased differentiation antigen expression, enhancedMHCclassI and II surface expression, and increases in other immunologi-cally relevant costimulatory molecules such as CD80 and CD86(23–25). These changes lead to augmented antitumor responsesinmodels ofmurinemelanomaadoptive cell therapy (26).HDACinhibitors can also reduce "negative" cell populations, such asmyeloid-derived suppressor cells, and augment checkpoint block-ade therapies, such as PD-1 blockade (27).

Herein, we demonstrate a novel role for HDAC inhibitors inupregulating PD-L1 and PD-L2 inmelanoma cells in vitro and in aB16F10 mouse model in vivo. HDAC inhibition induced pro-longed PD-L1 expression in both human and mouse cell lines.In vitro, patient melanoma samples expressed high amounts ofPD-L1 and PD-L2 in a dose-dependent manner in response toHDAC inhibitors. As a result of HDAC inhibition, a more relaxedchromatin state at the promoter regions of PD-L1 and PD-L2, andresultant increased gene expression, were observed. Combiningthe pan-HDAC inhibitor LBH589 with PD-1 blockade resulted inreduced tumor burden and improved survival in amurine B16F10model of established tumor. Although PD-L1 and PD-L2 nega-tively orchestrate the immune response against tumor, the resultsherein demonstrate that combining PD-1 blockade with HDACinhibition overcomes this hindrance to immunotherapy andprovide a strong rationale for combinatorial therapy with theseagents for the treatment of melanoma.

Materials and MethodsCell lines and patient samples

The human melanoma cell lines WM793, WM983A, WM35,WM1366, and the murine cell line B16F10 were purchased fromthe ATCC. Mel-624 and Mel-888 were kindly provided by Dr.Shari Pilon-Thomas, and SkMel21 byDr. Keiran Smalley at H. LeeMoffitt CancerCenter (Tampa, FL).Human cell lineswere authen-ticated using short tandem repeat profiling within 6 monthsbefore article submission. Melanoma patient samples wereobtained from surgical biopsies (clinical trial MCC15375; IRB

approved protocol #106509; H. Lee Moffitt Cancer Center) andprovided by Dr. Amod Sarnaik (F. Lee Moffitt Cancer Center). Allcells were cultured in RPMI-1640, supplemented with 10% FBS,non-essential amino acids, penicillin, streptavidin, amphotericinB, and Mycozap.

Mouse modelsC57BL/6 mice were purchased from NCI Laboratories and

Charles River Laboratories, and housed at H. Lee Moffitt CancerCenter animal facility. For in vivo studies,micewere s.c. inoculatedwith 1 � 105 B16F10 melanoma cells. Assessment of tumorgrowth and survival was performed after i.p. administration of15 mg/kg of LBH589 three times a week (Monday, Wednesday,and Friday) alone or in combination with 3 mg/kg of PD-1blocking antibody from BioXCell twice weekly (Tuesday andThursday), for 3 weeks. Treatments started 7 days after B16F10inoculation. Dextrose 5% was used in the treatment controlgroup. Tumor volume was assessed by caliper measurement andcalculated by the formula (width2� length)/2. For analysis of PD-L1 and PD-L2 expression in vivo, mice were treated three timeswith 15 mg/kg of LBH589 or dextrose 5% 10 days after B16F10inoculation. Mice were euthanized and tumors were harvestedwithin 2 hours after the last treatment for flow-cytometry analysis.All animal studies were in agreement with protocols approved bythe IACUC at the University of South Florida.

HDAC inhibitorsLBH589was kindly provided byNovartis. MGCD0103,MS275,

PXD101, PCI34051, and ACY1215 were purchased from SelleckChemicals. DMSO-reconstituted HDAC inhibitors aliquots werestored at �80�C. For in vitro studies, stocks were diluted to finalconcentration immediately before use. For in vivo use, LBH589wasdissolved and sonicated in 5% dextrose.

Flow-cytometry analysesFor cell surface analyses, melanoma cells were treated with

HDAC inhibitors for 24, 48, or 72 hours, as indicated. Cells wereharvestedwith Accutase, washed, and resuspended in FACS buffer(PBS, 2 mmol/L EDTA, 2% FBS). Cells were stained with phy-coerythryn, fluorescein isothiocyanate, or allophycocyanin-con-jugated antibodies from eBioscienece against PD-L1 and PD-L2,for 30 minutes at 4�C. Cells were then washed, resuspended inFACS buffer containing DAPI (50 ng/mL), and immediatelyacquired using an LSR II flow cytometer from BD Biosciences.

Patient-derived melanoma cells were also verified by flowstaining with fluorescein isothiocyanate and alexa fluor 405–conjugated antibodies against S100 andMart-1, from Abcam andNovusbio, respectively. Intracellular staining was performedusing the transcription factor staining buffer set from eBioscience,according to the manufacturer's instructions. Analyses were per-formed using FlowJo software.

Western blot analysisCellswere lysedwith lysis buffer (1% SDS, 4MUrea, 100nmol/L

dithiothrietol in 100 nmol/L Tris) and sonicated on ice for 16min-utes of alternated on/off 30-second pulses. Lysates were mixed 5:1with gel loading buffer [0.2% (weight/volume)] bromophenolblue, 200 mmol/L DTT, and 20% glycerol and boiled for 15minutes. Samples were electrophoresed in a SDS-PAGE gel andtransferred to a nitrocellulosemembrane. Incubationwith primaryantibody was performed overnight at 4�C. Antibodies against

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acetylated histone 3, total histone 3, acetylated a-tubulin andb-actin were purchased from Cell Signaling Technology. Immuno-blots were incubated with appropriate IRDYE secondary antibodyfor 2 hours and developed using a LI-COR instrument.

Chromatin immunoprecipitationChromatin preparation was performed as described by Desai

and colleagues (28), adjusted for the number of cells for eachimmunoprecipitation, and substituted with a concentration of0.5 mmol/L EGTA for buffers containing this reagent. Briefly, 5�106 cells were treated for 2 hours with LBH589 12.5 nmol/L orDMSO control. Five micrograms of primary antibodies for acet-ylated histone 3 from Active Motif and rabbit control IgG fromThermo Fisher Scientific were used for each immunoprecipita-tion. After overnight antibody incubation, reactions were incu-bated for 2hours at 4�Cwith 50mLof proteinA/Gplus beads fromSanta Cruz Biotechnology. DNA purification was done by usingthe MiniElute PCR Purification Kit from Qiagen, following themanufacturer's instructions. Evaluation of the ChIP was per-formed by SYBRGreen-based quantitative real-time PCR fromBio-Rad Laboratories using a Bio-Rad CFX96 PCR instrument.ChIP primers were designed using NCBI-Blast and covered 1800base pairs upstream the start codon of PDL-1 and PDL-2 humangenes. Amplicons were between 60 and 150 base pairs. Primerswere as follows:

PDL-1 promoter region:

Fw 50-GGCAAATTCCGTTTGCCTCA-30 Rv 50-TCCTCCTA-GATGGCCTGGAT-30,Fw 50-GCTGGGCCCAAACCCTATT-30 Rv 50-TTTGGCAG-GAGCATGGAGTT-30,Fw 50-CTAGAAGTTCAGCGCGGGAT-30 Rv 50-GGCCCAA-GATGACAGACGAT-30,Fw 50-ATGGGTCTGCTGCTGACTTT-30 Rv 50-GGCGTCCC-CCTTTCTGATAA-30,Fw 50-GGGGGACGCCTTTCTGATAA-30 Rv 50-AAGCCAA-CATCTGAACGCAC-30,Fw 50-ACTGAAAGCTTCCGCCGATT-30 Rv 50-CCCAAGG-CAGCAAATCCAGT-30,Fw 50-AGGACGGAGGGTCTCTACAC-30 Rv 50-ATTGGCTC-TACTGCCCCCTA-30,Fw 50-GTAGGGAGCGTTGTTCCTCC-30 Rv 50-GTGTAGA-GACCCTCCGTCCT-30,Fw 50-TAGGGGGCAGTAGAGCCAAT-30 Rv 50-CAAAACT-GAATCGCGCCTGG-30;

PD-L2 promoter region:

Fw 50-CCTGGCACAGCACTAAGACA-30, Rv 50-CTTCCCCATT-GTCCCTGGAG-30,Fw 50-GGCAGCAGGAGAAGGATTGA-30, Rv 50-GCCCCACTA-TACCTTCAGGC-30,Fw 50-TGGCTGTTCATTTTGGTGGC-30, Rv 50-ATGAGGACT-TGCCACAGCTC-30,Fw 50-AAGGGTGGCCTACCTTCTCT-30, Rv 50-TCTGGGGCA-GGAGGACATTA-30.

Quantitative real-time PCRCells were lysed by TRizol from Invitrogen. RNA was isolated

from samples by a standard phenol-chloroform separation pro-tocol, and cDNA generated by an iScript kit from Bio-Rad.

Expression was assessed by quantitative real-time PCR using aSYBRGreen platform on a Bio-Rad CFX96 system. Resultant datawere assessed by Bio-Rad CFX software and calculated by theformula 2[�(DDCt)] for relative mRNA expression. Correlation ofmRNA levels with PD-L1 surface expression and gene acetylationwas calculated by the formula 2(�DCt), adjusted by �105. 18Sribosomal RNA was used as the reference gene in all experiments.Primers were designed using NCBI-Blast and are as follows:

PD-L1: Fw 50-TCCTGAGGAAAACCATACAGC-30, Rv 50-GATG-GCTCCCAGAATTACCA-30;18S: Fw 50-GTAACCCGTTGAACCCCATT-30, Rv 50-CCATCCA-ATCGGTAGTAGCG-30.

Statistical analysisThe statistical significance of PD-L1 and PD-L2 expression

was determined by unpaired, two-tailed, Student t test. Differencein tumor growth was evaluated by one-way ANOVA, at theindicated time points. Mouse survival was assessed by theKaplan–Meier survival analysis log-rank test. For correlation ofPD-L1 surface expression, gene acetylation, and gene-expressiondata, analyses of correlation significance, Pearson R2 values, andlinear regression were performed. All statistical analyses wereperformed using GraphPad Prism 6.0 software. Findings withP values less than 0.05 were considered statistically significant.

ResultsHDAC inhibitors upregulate expression of PD-L1 inmelanomacell lines

To determine the differential effects of HDAC inhibitors onmelanoma, B16F10 murine melanoma cells were treated in vitrowith pan and selective HDAC inhibitors for 2 or 24 hours at theindicated doses. Increases in acetylated histone 3 is a surrogate forclass I HDAC inhibition, whereas increased acetylated a-tubulinis a surrogate for inhibition of HDAC6 (29). At 2 hours oftreatment, both LBH589 (panobinostat) andMGCD0103 (moce-tinostat) increased acetylated histone 3, whereas MS275 (etino-stat) resulted in no observable increase (Fig. 1A). However, at 24hours of treatment, MS275-treated cells displayed higher acety-lated histone 3, LBH589-treated melanoma continued to displayincreased acetylation of histone 3, and MGCD0103 regressed tonear basal levels. Neither the selectiveHDAC6 inhibitor ACY1215(rocilinostat), nor the selective HDAC8 inhibitor PCI34051 hadany observable effect on acetylated histone 3 at 2 or 24 hours oftreatment. ACY1215-treated cells did display higher levels ofacetylated a-tubulin. At 2 hours of treatment, LBH589 alsoresulted in a less profound, but observable increase in acetylateda-tubulin. Total histone 3 and b-actin were used as loadingcontrols.

Using doses of HDAC inhibitors chosen for optimal specificity,a panel of murine and humanmelanoma cell lines was evaluatedfor PD-L1 expression after treatment. The growth phase of thetumor from which they are derived and the mutational status ofthe human cell lines tested are as follows: WM983A—verticalgrowth phase—mutations in BRAF and p53; WM793—verticalgrowth phase—mutations in BRAF, PTEN, and CDK4; WM35—radial growth phase—mutations in BRAF and PTEN; Mel-624—metastatic—mutation in BRAF; Mel-888—combination of threesubcutaneous lesions—mutation in BRAF; WM1366—verticalgrowth phase—mutations in NRAS; SkMel-21—metastatic—mutation in NRAS (30–34). The murine melanoma cell lines

HDAC Inhibition Upregulates PD-1 Ligands in Melanoma

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B16F10 andB78H1were also evaluated.Melanoma cell lineswerecultured in the presence of 10 nmol/L LBH589, 500 nmol/LMS275, 500 nmol/L MGCD0103, or DMSO as control. After72 hours, melanomas were disassociated from plates by Accutaseto preserve surface markers. Cells were stained and assessed byflow cytometry for expression of PD-L1 in live cells. Autofluor-

escence was determined by the use of fluorescence minus onecontrol. In all cell lines tested, the three HDAC inhibitors upre-gulated surface expression of PD-L1, though to varying degrees(Fig. 1B and Table 1). Although expression was elevated aboveautofluorescence in all evaluated cell lines, varying degrees ofbasal PD-L1 expression were seen. In addition, doubling the dose

Figure 1.HDAC inhibitors upregulate PD-L1 in melanoma. A, B16F10 melanoma cells were cultured for 2 and 24 hours in the presence of indicated HDAC inhibitors. Cells werewashed, lysed, and analyzed by immunoblotting for acetylated histone 3, total histone 3, acetylated a-tubulin, and b-actin. B, indicated melanoma celllines were treated with 500 nmol/L MS275 (red), 10 nmol/L LBH589 (orange), 500 nmol/L MGCD0103 (purple), or DMSO control (black) for 72 hours in vitroand PD-L1 expression was evaluated. C, indicated melanoma cell lines were plated and treated with 500 nmol/L MS275 (triangles), 10 nmol/L LBH589(squares), or 500 nmol/L MGCD0103 (diamonds) at 96, 72, 48, or 24 hours before evaluation of PD-L1. Expression of DMSO-treated cells was graphed as zero-hourtreatment. All values are graphed as mean fluorescence intensity (MFI) with autofluorescence values subtracted. Results shown are representative of two to threeindependent experiments.






of inhibitor used for LBH589, MS275, and MGCD0103 furtherelevated the expression of PD-L1 (Supplementary Fig. S1).

In addition to evaluating the effects of HDAC inhibitors,the impact of DNA methylation inhibitors on melanoma PD-L1expression was evaluated. At 48 hours of treatment with 5 mmol/Lof azacitidine or 500 nmol/L of decitabine, bothMel-624 andMel-888 experienced significant upregulation of PD-L1 and PD-L2surface expression (Supplementary Fig. S2). However, 1 mmol/Lof RG108 did not produce a change in either marker.

Upregulation of PD-L1 by HDAC inhibition is durableTo determine the kinetics of the observed PD-L1 upregulation,

the melanoma cell lines WM983A, WM793, and B16F10 were

plated and treated for the indicated durations with LBH589,MS275, or MGCD0103 at the indicated doses and harvested foranalysis as described above. Themean fluorescent intensity (MFI)of PD-L1 expression was determined (Fig. 1C). Subtracting cellline–specific autofluorescence provided a zero value indicating noexpressionof PD-L1. Initial, zero-hour expressionwasdeterminedby DMSO-treated controls' MFI. Detectable upregulation of PD-L1 was observed as early as 24 hours in all three cell lines. Peakexpression was seen as early as 48 hours or as late as 96 hours,depending upon the inhibitor and the cell line. In addition, thedegree of upregulation induced by the individual HDAC inhibi-tors differed. In B16F10, MS275 induced the least amount ofupregulation, with LBH589 intermediate and MGCD0103 themost robust. Indeed,MGCD0103 treatment was still increasing atthe 96-hour time point. In contrast with B16F10,WM983APD-L1expression had the highest degree of upregulation in response toMS275. WM793 was most affected by LBH589, with peak expres-sion at 72 hours.

HDAC inhibitorswith specificity for class IHDACs increase PD-L1 and PD-L2 expression on patient melanomas in a dose-dependent manner

To expand on these findings, several primary human mela-nomas were treated with an expanded panel of HDAC inhibi-tors. Patient melanomas were expanded in vitro and verified forexpression of the melanoma markers Mart1 and S100 (Sup-plementary Fig. S3; ref. 35). Cultured cells were treated for 24hours with the indicated HDAC inhibitors or DMSO controls.After 24 hours, media and inhibitors were removed, cells werewashed twice, and fresh media added to cultures. After anadditional 48 hours (72 hours after the initial treatment), cellswere harvested as described above and stained for PD-L1 andPD-L2; data were acquired by flow cytometry. For all cell lines,DMSO controls were run in triplicate. AutofluorescenceMFI values were subtracted and adjusted MFI values graphed(Fig. 2). As before, PD-L1 expression was enhanced withLBH589, MS275, and MGCD0103 treatment (three represen-tative patient samples in Fig. 2A). PDX101 (belinostat), a pan-HDAC inhibitor, similarly upregulated PD-L1 expression. Fur-thermore, these four inhibitors had a clear dose-dependenteffect, with higher concentrations resulting in even greaterexpression of PD-L1. Of the inhibitors evaluated, LBH589had the greatest ability to enhance PD-L1 expression, withthe highest levels achieved at 20 times less than the concen-tration of the other evaluated inhibitors. Unlike these fouragents, ACY1215, Nexturastat A, and PCI34051 produced nochanges in PD-L1 expression at the concentrations evaluated.ACY1215 and Nexturastat A are isotype selective for HDAC6,while PCI34051 is selective for HDAC8, and the concentrationsevaluated were chosen to remain within their described speci-ficities (36, 37). These results suggest that the observed upre-gulation of PD-L1 is confined to inhibition of theclass I HDAC1, HDAC2, and/or HDAC3. Similar to PD-L1,PD-L2 expression was also enhanced by LBH589, MS275,MGCD0103, and PDX101, but not ACY1215, Nexturastat A,or PCI34051 (Fig. 2B). PD-L2 displayed less consistent, but stilllargely dose-dependent upregulation after HDAC inhibition.

PD-L1 and PD-L2 upregulation occurs in vivoThe ability of HDAC inhibitors to upregulate PD-1 ligands in

vivowas investigated using a B16F10murine model. LBH589 was

Table 1. PD-L1 expression in melanoma cell lines as a result of HDAC inhibition

Cell line TreatmentPD-L1MFI

Change overDMSO (%)

WM983A Autofluorescence 328 NADMSO 386 NALBH589 (10 nmol/L) 835 216MS275 (500 nmol/L) 546 141MGCD0103 (500 nmol/L) 1,151 298

WM793 Autofluorescence 1,568 NADMSO 1,744 NALBH589 (10 nmol/L) 2,361 135MS275 (500 nmol/L) 2,730 157MGCD0103 (500 nmol/L) 2,108 121

B78H1 Autofluorescence 651 NADMSO 9,044 NALBH589 (10 nmol/L) 13,839 153MS275 (500 nmol/L) 13,051 144MGCD0103 (500 nmol/L) 18,364 203

SkMel21 Autofluorescence 1,404 NADMSO 3,686 NALBH589 (10 nmol/L) 10,616 288MS275 (500 nmol/L) 16,508 448MGCD0103 (500 nmol/L) 7,171 195

WM35 Autofluorescence 698 NADMSO 1,095 NALBH589 (10 nmol/L) 1,654 151MS275 (500 nmol/L) 1,693 155MGCD0103 (500 nmol/L) 1,261 115

WM1366 Autofluorescence 648 NADMSO 4,788 NALBH589 (10 nmol/L) 8,184 171MS275 (500 nmol/L) 6,999 146MGCD0103 (500 nmol/L) 10,169 212

B16 Autofluorescence 206 NADMSO 1,297 NALBH589 (10 nmol/L) 4,139 319MS275 (500 nmol/L) 2,480 191MGCD0103 (500 nmol/L) 7,350 567

624 Autofluorescence 1,337 NADMSO 4,241 NALBH589 (10 nmol/L) 6,211 146MS275 (500 nmol/L) 6,446 152MGCD0103 (500 nmol/L) 7,142 168

888 Autofluorescence 1,027 NADMSO 2,097 NALBH589 (10 nmol/L) 2,999 143MS275 (500 nmol/L) 3,608 172MGCD0103 (500 nmol/L) 3,281 156

NOTE: Mean fluorescence intensity (MFI) and the percentage (%) of changeover DMSO control are shown for various melanoma cell lines treated for72 hours with LBH589, MS275, and MGCD0103 HDAC inhibitors, at indicatedconcentrations.Abbreviation: NA, not applicable.






chosen for continued evaluation due to its high potency. FiveC57BL/6 mice per group were s.c. injected with 105 B16F10melanoma cells. Tumors were allowed to grow, and on day 10,once tumors became visible, treatment with LBH589 (15 mg/kg)or 5% dextrose control was given for 3 consecutive days. On thethird day of treatment, tumors were excised, physically disasso-ciatedby repeatedpassage of tumors througha70-mmsterilefilter,and stained. Expression of PD-L1 (Fig. 3A) andPD-L2 (Fig. 3B) onviable, CD45� cells was assessed by flow cytometry and reportedas the average MFI � the SEM. LBH589 treatment resulted in asignificant increase of PD-L1 expression compared with control-treated tumors. Indeed, the average approximately doubled thePD-L1 expression of the dextrose control. Likewise, a significantincrease in PD-L2 expression in the LBH589-treated group com-pared with the control was seen (P < 0.01). PD-L2 expression,though enhanced,wasupregulated to a lessermagnitude thanPD-L1. Basal PD-L2 expression was also low, relative to PD-L1expression.

HDAC inhibitors relax the chromatin state at thePD-L1 andPD-L2 gene promoters

To determine potential changes in the chromatin state byHDAC inhibition in the PD-L1 and PD-L2 genes, the histoneacetylation changes resulting from treatment were analyzed.WM983A melanoma cells were treated with DMSO control or12.5 nmol/L LBH589 for 2 hours, at which time cells were fixed.Chromatin immunoprecipitation (ChIP) for pan-acetylated his-tone 3 was performed (Fig. 4A). LBH589-treated cells displayedmore acetylation in the promoter region of the PD-L1 gene. Peakacetylation in the LBH589-treated samplewasobserved at approx-imately 455 base pairs upstream of the first exon of the PD-L1gene, with acetylation changes tapering off at approximately1,700 base pairs toward the 50 end and into the gene region. ThePD-L2 gene had low basal acetylation, which was marginallyincreased after LBH589 treatment (Fig. 4B). To expand uponthesefindings, additionalmelanoma cell lineswere also evaluatedby ChIP in a similar fashion as above (Fig. 4C). All melanomas

Figure 2.Inhibition of class I HDACs increasesPD-L1 andPD-L2 expressions in patientmelanomas in a dose-dependentmanner. Patientmelanomasobtained frombiopsies andexpanded in culture were plated and treated with indicated HDAC inhibitors and concentrations for 24 hours. Cells were then washed and cultured for afurther 48 hours. At 72 hours past initial treatment, melanomas were evaluated for expression of PD-L1 (A) and PD-L2 (B). DMSO controls were run intriplicate. MFI values are graphed with autofluorescence values subtracted.






tested displayed higher histone acetylation at the PD-L1promoter after LBH589 treatment. Likewise, higher histoneacetylation at the PD-L2 gene, resulting from LBH589 treatment,was also seen (Fig. 4D).

HDAC inhibitor–mediated chromatin acetylation leads toincreases in PD-L1 gene expression

As increased histone acetylation is indicative of a relaxedchromatin structure and enhanced gene expression, the impactof the observed increased acetylation of histone 3 on PD-L1gene expression was evaluated. To determine the kinetics of PD-L1 expression after inhibiting HDAC, WM983A cells weretreated with 12.5 nmol/L LBH589 for 6, 14, 24, or 48 hours.Expression of PD-L1 was elevated as early as 6 hours aftertreatment, and expression continued to increase up to or past48 hours (Fig. 5A). To further validate these findings, addi-tional melanoma cell lines were also investigated for PD-L1expression after 6 hours of LBH589 treatment (Fig. 5B). Expres-sion of PD-L1 was upregulated after LBH589 treatment, tovarying degrees, in all cell lines.

As histone acetylation is a known mechanism governing genetranscriptional activity, and thus protein expression, the associ-ation of these three observed values was explored. To this end, theDMSO treatment MFI values (minus autofluorescence) of PD-L1surface expression from Fig. 1B, PD-L1 promoter–associatedacetylated histone 3-fold enrichment values from Fig. 4C, andPD-L1 mRNA expression from Fig. 5B were evaluated for corre-lation. Correlations were found between PD-L1 surface expres-sion and acetylation (R2¼ 0.5958), PD-L1 surface expression andmRNA expression (R2¼ 0.5649), and PD-L1 gene acetylation andmRNA expression (R2 ¼ 0.861; Fig. 5C–E).

CombiningHDAC inhibitionwith PD-1 blockade delays tumorprogression and enhances survival

Given the previous demonstration of the anti-melanomaeffects of HDAC inhibitors and the observed upregulation ofPD-1 ligands, the therapeutic efficacy of disrupting the PD-1inhibitory axis in combination with HDAC inhibition wasexplored. To this end, C57BL/6 mice were injected s.c. withB16F10 melanoma cells. Seven days later, 10 mice per groupwere treated with LBH589 (15 mg/kg), PD-1–blocking antibody(3 mg/kg), a combination of both agents or a dextrose control.This dose of LBH589 has been previously described (23), and didnot affect mouse weight, a surrogate of toxicity (Supplementary

Fig. S4). Mice receiving treatment with a combination of PD-1blockade and LBH589 had significantly (P < 0.05) less tumorprogression than control-treated mice (Fig. 6A). Although nei-ther of the single-agent treatment groups achieved statisticalsignificance, LBH589-treated mice showed a trend toward low-er tumor volumes, in agreement with our previous publication(23). PD-1 blockade as a single agent achieved no discernibledifference in tumor progression versus control. Combinationtherapy also significantly improved survival (P < 0.05) overcontrol-treated mice (Fig. 6B). Median survival times were asfollows: dextrose, 29 days; LBH589, 34.5 days; PD-1 blockade,30.5 days; and combination therapy >37 days.

DiscussionThe results presented herein demonstrate the ability of

HDAC inhibitors of class I HDACs to increase the expressionof the PD-1 ligands, PD-L1 and PD-L2, in vitro and in vivo onmelanoma cells. This enhanced expression is sustained androbust in the case of PD-L1. Furthermore, this upregulationwas seen in all melanoma cell lines and patient samples tested,regardless of mutational status. An increased histone acetyla-tion at the promoter regions of the PD-L1 and PD-L2 genes wasassociated with increased PD-1 ligand expression. Accompa-nying the increased histone acetylation, an increase in PD-L1mRNA was seen, linking the enhanced surface expression withhistone acetylation changes. Supporting this model, correla-tions between the basal PD-L1 surface expression, mRNAexpression, and promoter acetylation of evaluated cell lineswere observed. Compared with the PD-L1 gene, the PD-L2gene had lower basal histone acetylation. This may explainlow basal PD-L2 expression. Although LBH589 treatmentincreased acetylation levels in the PD-L2 gene, the increasewas mild. Collectively, these data support a model in whichHDAC inhibition increases chromatin relaxation, enhancinggene expression and resultantly PD-1 ligand surface expressionon melanoma cells.

Intriguingly, two DNA methyltransferase inhibitors, azaciti-dine and decitabine, were also able to augment PD-L1 and PD-L2 expression in both the melanoma cell lines assessed. Contraryto these results, a third methyltransferase inhibitor, RG108, didnot alter PD-1 ligand expression. Whether increased doses wouldresult in detectable increases in PD-L1 or PD-L2 remains to beinvestigated. Regardless, these data are provocative and raiseseveral questions. For example, is the upregulation by DNA

Figure 3.HDAC inhibitors upregulate PD-L1 andPD-L2 expression in vivo. C57BL/6micewere inoculated s.c. with B16F10melanoma. When tumors were visible,10 days after inoculation, mice receivedtreatment with 15 mg/kg LBH589 ordextrose control (5 mice/group) for3 consecutive days. On the third day oftreatment, tumors were harvested.A, PD-L1 and B, PD-L2 expressionwere evaluated by flow cytometry;� , P < 0.05; �� , P < 0.01.






methyltransferase inhibitors through a related or independentpathway as that of HDAC inhibitors detailed in this study?Supporting a hypothesis of relatedmechanisms,methylatedDNAis known to recruit HDACs, thereby repressing transcription (38).In addition, HDACs inhibitors have been characterized to down-regulate DNA methyltransferase activity (39). Studies are underway to address these possibilities.

In this study, all cell lines and primary melanomas evaluatedshowed upregulation of PD-L1 in response to class I HDACinhibition. Furthermore, all cell lines had detectable PD-L1expression. Therefore, whether basal expression of PD-L1 isnecessary for the described upregulation remains to be investi-gated. As HDAC inhibition may only increase the transcriptionalaccessibility of the PD-L1 promoter, reliance on already function-al transcriptional machinery (e.g., STATs) allowing basal expres-sion may be necessary for the observed upregulation. Of the celllines investigated, WM793 expressed minimal PD-L1 surface

protein in the basal state and had the lowest basal amount ofhistone 3 acetylation at the PD-L1 gene. Although this basalacetylation and expression were minimally above background,HDAC inhibition still upregulated expression. The B78H1 linehad high PD-L1 expression, but had comparatively less upregula-tion of PD-L1 withHDAC inhibition compared with B16F10. It ispostulated that this may be the result of several previouslydescribed dysfunctional gene transcripts in B78H1 (40). Theseobservations raise the question of what differences regulating thebasal expression of PD-L1 exist in differentmelanomas. Althoughbeyond the scope of this study, these questions are important toaddress in the future for a more complete understanding of thebasic biology of immune–tumor interactions and the obviousclinical implications, and are currently being explored.

Although these results demonstrated the ability of HDACinhibition to upregulate PD-1 ligand expression in melanoma,the particular HDAC(s) governing the expression of these

Figure 4.HDAC inhibition increases histone acetylation at the PD-L1 and PD-L2 promoters. Indicated melanoma cell lines were treated in vitro for 2 hours with12.5 nmol/L LBH589 (squares) or DMSO control (circles). Cells were then fixed and chromatin immunoprecipitated for acetylated histone 3 or IgG control. DNApulldown was quantified by qRT-PCR. Fold enrichment over corresponding IgG pulldown at the PD-L1 (A) and PD-L2 (B) gene regions for WM983A aregraphed. Results shownare representative of two independent experiments. Five other cell lineswere assessed once for acetylation at the�455gene region of PD-L1(C) and þ307 gene region of PD-L2 (D). For all graphs, error bars are representative of technical replicates.






molecules remain to be fully elucidated. Although LBH589 is apan-HDAC inhibitor, with a broad specificity against all HDACs,mocetinostat (MGCD0103) and etinostat (MS275) are class IHDAC inhibitors, most potent against HDACs 1, 2, 3, and 11 andHDACs1 and3, respectively (41, 42). These agents all upregulatedPD-1 ligand expression, whereas HDAC6- and HDAC8-specificinhibitors could not. These data would suggest that HDAC1,HDAC2, HDAC3, or potentially a combination of these epige-netic regulators are responsible for maintaining the histone acet-ylation status of the PD-L1 and PD-L2 genes, thereby regulatingthe expression of these molecules. Indeed, these three class IHDACs are largely localized to thenucleus; the remaining classicalHDACs can be found in the cytoplasm (22). On the basis of thisknowledge, it is hypothesized that HDAC1, HDAC2, and/orHDAC3 control the histone acetylation of these genes, therebyregulating, in part, their transcriptional activity. This hypothesis iscompatible with the data presented in this study, but remains tobe further tested. Indeed, future identification of the specifics ofwhich HDAC and possibly other epigenetic modifiers may allowfor the use of selective agents to better alter the immune response,not only in malignancy, but also in autoimmunity.

Clinically, expression of PD-L1 in tumors revealed by immu-nohistochemical staining is associated with patient response toPD-1 blockade (43). This likely results from PD-L1 expressionreflecting an active immune response in the tumor microenvi-ronment. Lackof expressionof PD-L1by tumorsmay result fromalack of proinflammatory immune infiltrate, and consequently alack of T cells generating cytokines that influence PD-L1 expres-sion. Indeed, PD-L1 and PD-L2 expression are upregulated in

response to the proinflammatory cytokines IFNg and TNF (44).Under this model, the upregulation of PD-1 ligands by HDACinhibition, independent of increased immune infiltration,represents an undesirable effect among the previously demon-strated immune-enhancing effects (e.g., increased MHC andantigen expression) of HDAC inhibitors. Consequently, theblockade of this pathway may increase the efficacy of HDACinhibitor treatment as an immunotherapeutic strategy.

Therapeutically, combining the HDAC inhibitor LBH589 withPD-1 blockade in vivo enhanced antitumor activity comparedwitheither agent alone, resulting in a delay in tumor progression andan increase in OS in murine melanoma. Previously, it was shownthat LBH589 as a single agent increases the survival time ofB16F10 tumor–bearing, immunocompetent mice. However, thiseffect was lost on immunodeficient mice (23). A similar increasein survival with LBH589 as a single agent in immunocompetentmice was seen in this study. However, in the experiments pre-sented herein using the B16F10 model, PD-1 blockade as a singleagent did not prolong survival compared with control treatments.Curran and colleagues (45) showed that PD-1 blockade hadminimal effects in murine B16F10 models unless combined withvaccination, a result reminiscent of that seen withmurine CTLA-4blockade (46). Intriguingly, the efficacy of PD-1 blockade wasenhanced with LBH589 treatment without the need for vaccina-tion. This may be the result of the ability of HDAC inhibitors toaugment MHC and differentiation antigen expression in mela-noma, achieving a superior ability to activate T cells (23).

Collectively, these results add to the growing body of datademonstrating that HDAC inhibitors may alter the immune

Figure 5.PD-L1mRNA expression increased following HDAC inhibition, correlatingwith protein expression and gene acetylation. A,WM983A cells were treatedwith DMSOor12.5 nmol/L LBH589 for indicated time points. Cells were assessed by qRT-PCR for PD-L1 expression. B, indicated cell lines were treated with DMSO or12.5 nmol/L LBH589 for 6 hours and subsequently assessed by qRT-PCR for PD-L1 expression. For all graphs, error bars are representative of technical replicates.Correlations of PD-L1 surface expression versus gene acetylation (C), PD-L1 surface expression versus gene expression (D), and PD-L1 gene acetylationversus gene expression (E) were assessed for various melanoma cell lines at basal state (DMSO control). Acetylated H3 was graphed as fold enrichment overcorresponding IgG pulldown at the �455 region of the PD-L1 gene. Gene expression was determined by qRT-PCR and calculated as fold units relativeto 18S endogenous ribosomal RNA. Flow-cytometry analysis of PD-L1 surface expression was indicated as MFI.






landscape of malignant cells, through changes in expression ofcostimulatory molecules, MHC and tumor antigens, as well ascytokine production by tumor cells. Likewise, these data furtherhighlight the intimate connection between epigenetics andimmune regulation. Although the results of this study raise manyquestions about the basic biology of PD-1 ligand expression inmelanoma, they also demonstrate clinical relevance for theimmune effects of HDAC inhibitors and provide a rationale forthe clinical evaluation of PD-1 blockade in combination withHDAC inhibition.

Disclosure of Potential Conflicts of InterestJ.Weber has received honoraria from the speakers bureau and is a consultant/

advisory board member for Bristol-Myers Squibb. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: D.M. Woods, A.L. Sodr�e, E.M. SotomayorDevelopment of methodology: D.M. Woods, A.L. Sodr�e, A. Villagra,E.M. SotomayorAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D.M. Woods, A.L. Sodr�e, A. Sarnaik, E.M. Sotomayor

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):D.M.Woods, A.L. Sodr�e, A. Villagra, E.M. Sotomayor,J. WeberWriting, review, and/or revision of the manuscript: D.M. Woods, A.L. Sodr�e,A. Villagra, E.M. Sotomayor, J. WeberAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.M. Woods, A.L. Sodr�eStudy supervision: D.M. Woods, A.L. Sodr�e, E.M. Sotomayor, J. Weber

AcknowledgmentsThe authors' appreciation is extended to Jodi Kroeger and the Flow Cyto-

metry Core at H. Lee Moffitt Cancer Center (Tampa, FL) for their expertise andassistance in flow cytometry. The authors also thank Susan Deng for herinvaluable expertise in immunoblotting.

Grant SupportThis research was supported by a National Institutes of Health Skin SPORE

grant (IH/NCI P50 CA168536-01).The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 19, 2015; revised June 19, 2015; accepted July 14, 2015;published OnlineFirst August 21, 2015.

References1. In:Howlader N NA, Krapcho M, Garshell J, Miller D, Altekruse SF, Kosary

CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ,Cronin KA (editors). SEER Cancer Statistics Review, 1975–2011. 2014;Available from: http://seer.cancer.gov/csr/1975_2011/.

2. CostanzaME,Nathanson L, CostelloWG,Wolter J, Brunk SF, Colsky J, et al.Results of a randomized study comparing DTIC with TIC mustard inmalignant melanoma. Cancer 1976;37:1654–9.

3. Patel PM, Suciu S, Mortier L, Kruit WH, Robert C, Schadendorf D, et al.Extended schedule, escalated dose temozolomide versus dacarbazine instage IV melanoma: final results of a randomised phase III study (EORTC18032). Eur J Cancer 2011;47:1476–83.

4. Hodi FS,O'Day SJ,McDermott DF,Weber RW, Sosman JA,Haanen JB, et al.Improved survivalwith ipilimumab in patients withmetastaticmelanoma.N Engl J Med 2010;363:711–23.

5. Hamid O, Robert C, Daud A, Hodi FS, HwuWJ, Kefford R, et al. Safety andtumor responses with lambrolizumab (anti-PD-1) in melanoma. N EnglJ Med 2013;369:134–44.

6. Iwai Y, Ishida M, Tanaka Y, Okazaki T, Honjo T, Minato N. Involvement ofPD-L1 on tumor cells in the escape from host immune system and tumorimmunotherapy by PD-L1 blockade. Proc Natl Acad Sci U S A 2002;99:12293–7.

7. Latchman Y, Wood CR, Chernova T, Chaudhary D, Borde M, Chernova I,et al. PD-L2 is a second ligand for PD-1 and inhibits T-cell activation. NatImmunol 2001;2:261–8.

8. Sheppard KA, Fitz LJ, Lee JM, Benander C, George JA, Wooters J, et al. PD-1inhibits T-cell receptor induced phosphorylation of the ZAP70/CD3zetasignalosome and downstream signaling to PKCtheta. FEBS Lett 2004;574:37–41.

Figure 6.Combining HDAC inhibition with PD-1 blockade in vivo results in delayed tumor growth and enhanced survival. C57BL/6 mice were inoculated s.c. withB16F10 melanoma. Seven days after inoculation, mice began treatment with LBH589 (15 mg/kg, triangles; Monday, Wednesday, and Friday), PD-1–blockingantibody (3 mg/kg, squares; Tuesday and Thursday), a combination of these agents (diamonds) or dextrose control (circles) for 3 weeks. A, tumor growthwas measured; and B, survival monitored. The log-rank test of survival curve differences was P < 0.05. Ten mice were assessed per group and resultsshown are representative of two independent experiments; � , P < 0.05.






9. Parry RV, Chemnitz JM, Frauwirth KA, Lanfranco AR, Braunstein I, Kobaya-shi SV, et al. CTLA-4 and PD-1 receptors inhibit T-cell activation by distinctmechanisms. Mol Cell Biol 2005;25:9543–53.

10. Dong H, Strome SE, Salomao DR, Tamura H, Hirano F, Flies DB, et al.Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mecha-nism of immune evasion. Nat Med 2002;8:793–800.

11. Chemnitz JM, Parry RV, Nichols KE, June CH, Riley JL. SHP-1 and SHP-2associate with immunoreceptor tyrosine-based switch motif of pro-grammed death 1 upon primary human T cell stimulation, but onlyreceptor ligation prevents T-cell activation. J Immunol 2004;173:945–54.

12. Latchman YE, Liang SC, Wu Y, Chernova T, Sobel RA, KlemmM, et al. PD-L1-deficient mice show that PD-L1 on T cells, antigen-presenting cells, andhost tissues negatively regulates T cells. Proc Natl Acad Sci U S A 2004;101:10691–6.

13. KeirME, Liang SC,Guleria I, LatchmanYE,QipoA,Albacker LA, et al. Tissueexpression of PD-L1 mediates peripheral T-cell tolerance. J Exp Med2006;203:883–95.

14. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al.Colocalization of inflammatory response with B7-h1 expression in humanmelanocytic lesions supports an adaptive resistance mechanism ofimmune escape. Sci Transl Med 2012;4:127ra37.

15. Saunders PA, Hendrycks VR, Lidinsky WA, Woods ML. PD-L2:PD-1involvement in T-cell proliferation, cytokine production, and integrin-mediated adhesion. Eur J Immunol 2005;35:3561–9.

16. Yamazaki T, AkibaH, IwaiH,MatsudaH,AokiM, TannoY, et al. Expressionof programmed death 1 ligands by murine T cells and APC. J Immunol2002;169:5538–45.

17. Topalian SL, Drake CG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1)pathway to activate anti-tumor immunity. Curr Opin Immunol 2012;24:207–12.

18. Topalian SL, Sznol M, McDermott DF, Kluger HM, Carvajal RD, SharfmanWH, et al. Survival, durable tumor remission, and long-term safety inpatients with advanced melanoma receiving nivolumab. J Clin Oncol2014;32:1020–30.

19. Brahmer JR, Tykodi SS, ChowLQ,HwuWJ, Topalian SL,HwuP, et al. Safetyand activity of anti-PD-L1 antibody in patients with advanced cancer.N Engl J Med 2012;366:2455–65.

20. Taube JM, Klein A, Brahmer JR, Xu H, Pan X, Kim JH, et al. Association ofPD-1, PD-1 ligands, and other features of the tumor immune microenvi-ronment with response to anti-PD-1 therapy. Clin Cancer Res 2014;20:5064–74.

21. Weber JS, Kudchadkar RR, Yu B, Gallenstein D, Horak CE, Inzunza HD,et al. Safety, efficacy, and biomarkers of nivolumab with vaccine inipilimumab-refractory or -naive melanoma. J Clin Oncol 2013;31:4311–8.

22. DelcuveGP, KhanDH,Davie JR. Roles of histone deacetylases in epigeneticregulation: emerging paradigms from studies with inhibitors. Clin Epige-netics 2012;4:5.

23. Woods DM,Woan K, Cheng F, Wang H, Perez-Villarroel P, Lee C, et al. Theantimelanoma activity of the histone deacetylase inhibitor panobinostat(LBH589) is mediated by direct tumor cytotoxicity and increased tumorimmunogenicity. Melanoma Res 2013;23:341–8.

24. Khan AN, Tomasi TB. Histone deacetylase regulation of immune geneexpression in tumor cells. Immunol Res 2008;40:164–78.

25. MagnerWJ, KazimAL, Stewart C, RomanoMA, CatalanoG,GrandeC, et al.Activation of MHC class I, II, and CD40 gene expression by histonedeacetylase inhibitors. J Immunol 2000;165:7017–24.

26. Vo DD PR, Begley JL, Donahue TR, Morris LF, Bruhn KW, de la Rocha P,et al. Enhanced antitumor activity induced by adoptive T-cell transfer andadjunctive use of the histone deacetylase inhibitor LAQ824. Cancer Res2009;69:8693–9.

27. Kim K, Skora AD, Li Z, Liu Q, Tam AJ, Blosser RL, et al. Eradication ofmetastatic mouse cancers resistant to immune checkpoint blockade bysuppression of myeloid-derived cells. Proc Natl Acad Sci U S A 2014;111:11774–9.

28. Desai S, Bolick SC,MaurinM,Wright KL. PU.1 regulates positive regulatorydomain I-binding factor 1/Blimp-1 transcription in lymphoma cells.J Immunol 2009;183:5778–87.

29. Dokmanovic M, Clarke C, Marks PA. Histone deacetylase inhibitors:overview and perspectives. Mol Cancer Res 2007;5:981–9.

30. Tsao H, Goel V, Wu H, Yang G, Haluska FG. Genetic interaction betweenNRAS and BRAFmutations and PTEN/MMAC1 inactivation inmelanoma.J Invest Dermatol 2004;122:337–41.

31. Tanami H, Imoto I, Hirasawa A, Yuki Y, Sonoda I, Inoue J, et al. Involve-ment of overexpressed wild-type BRAF in the growth of malignant mel-anoma cell lines. Oncogene 2004;23:8796–804.

32. Herlyn M. Melanoma cell lines grouped for BRAF/-N-ras/PTEN/CDk4mutations. The Wistar Institute 2015.

33. Herlyn M, Thurin J, Balaban G, Bennicelli JL, Herlyn D, Elder DE, et al.Characteristics of cultured human melanocytes isolated from differentstages of tumor progression. Cancer Res 1985;45:5670–6.

34. Rivoltini L, Barracchini KC, Viggiano V, Kawakami Y, Smith A, Mixon A,et al. Quantitative correlation between HLA class I allele expression andrecognition ofmelanoma cells by antigen-specific cytotoxic T lymphocytes.Cancer Res 1995;55:3149–57.

35. Shidham VB, Qi DY, Acker S, Kampalath B, Chang CC, George V, et al.Evaluation of micrometastases in sentinel lymph nodes of cutaneousmelanoma: higher diagnostic accuracy with Melan-A and MART-1compared with S-100 protein and HMB-45. Am J Surg Pathol 2001;25:1039–46.

36. Santo L, Hideshima T, Kung AL, Tseng JC, Tamang D, Yang M, et al.Preclinical activity, pharmacodynamic, and pharmacokinetic properties ofa selective HDAC6 inhibitor, ACY-1215, in combination with bortezomibin multiple myeloma. Blood 2012;119:2579–89.

37. Balasubramanian S, Ramos J, Luo W, Sirisawad M, Verner E, Buggy JJ. Anovel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051induces apoptosis in T-cell lymphomas. Leukemia 2008;22:1026–34.

38. Jones PL, Veenstra GJ, Wade PA, Vermaak D, Kass SU, Landsberger N, et al.Methylated DNA and MeCP2 recruit histone deacetylase to repress tran-scription. Nat Genet 1998;19:187–91.

39. Xiong Y, Dowdy SC, Podratz KC, Jin F, Attewell JR, Eberhardt NL, et al.Histone deacetylase inhibitors decrease DNA methyltransferase-3B mes-senger RNA stability and down-regulate de novo DNA methyltransferaseactivity in human endometrial cells. Cancer Res 2005;65:2684–9.

40. Chiang EY, HensonM, Stroynowski I. Correction of defects responsible forimpaired Qa-2 class Ib MHC expression on melanoma cells protects micefrom tumor growth. J Immunol 2003;170:4515–23.

41. Arts J, KingP,MarienA, FlorenW,BelienA, Janssen L, et al. JNJ-26481585, anovel "second-generation" oral histone deacetylase inhibitor, showsbroad-spectrum preclinical antitumoral activity. Clin Cancer Res 2009;15:6841–51.

42. Beckers T, Burkhardt C, Wieland H, Gimmnich P, Ciossek T, Maier T, et al.Distinct pharmacological properties of second generation HDAC inhibi-tors with the benzamide or hydroxamate head group. Int J Cancer 2007;121:1138–48.

43. TopalianSL,Hodi FS, Brahmer JR,Gettinger SN, SmithDC,McDermottDF,et al. Safety, activity, and immune correlates of anti-PD-1 antibody incancer. N Engl J Med 2012;366:2443–54.

44. Rodig N, Ryan T, Allen JA, Pang H, Grabie N, Chernova T, et al. Endothelialexpression of PD-L1 and PD-L2 down-regulates CD8þ T cell activation andcytolysis. Eur J Immunol 2003;33:3117–26.

45. Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4 combi-nation blockade expands infiltrating T cells and reduces regulatory T andmyeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A2010;107:4275–80.

46. van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy ofB16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4(CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and meta-static tumors accompanied by autoimmune depigmentation. J Exp Med1999;190:355–66.






2015;3:1375-1385. Published OnlineFirst August 21, 2015.Cancer Immunol Res David M. Woods, Andressa L. Sodré, Alejandro Villagra, et al. Augments Immunotherapy with PD-1 BlockadeHDAC Inhibition Upregulates PD-1 Ligands in Melanoma and

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