HDAC3InhibitionUpregulatesPD-L1Expressionin B-Cell ... · Small Molecule Therapeutics...

Small Molecule Therapeutics

HDAC3 InhibitionUpregulatesPD-L1Expression inB-Cell Lymphomas and Augments the Efficacy ofAnti–PD-L1 TherapySiyu Deng, Qianwen Hu, Heng Zhang, Fang Yang, Cheng Peng, and Chuanxin Huang

Abstract

Programmed cell-death protein 1 (PD-1) and pro-grammed death-ligand 1 (PD-L1) pathway blockade is apromising therapy for the treatment of advanced cancers,including B-cell lymphoma. The clinical response to PD-1/PD-L1 immunotherapy correlates with PD-L1 levels ontumor cells and other cells in the tumor microenvironment.Hence, it is important to understand the molecular mechan-isms that regulate PD-L1 expression. Here, we reportthat histone deacetylase 3 (HDAC3) is a crucial repressorof PD-L1 transcription in B-cell lymphoma. Pan-HDACs orselective HDAC3 inhibitors could rapidly increase histoneacetylation and recruitment of bromodomain protein BRD4at the promoter region of PD-L1 gene, leading to activation

of its transcription. Mechanically, HDAC3 and its putativeassociated corepressor SMRT were recruited to the PD-L1promoter by the transcriptional repressor BCL6. In addition,HDAC3 inhibition reduced DNA methyltransferase 1protein levels to indirectly activate PD-L1 transcription.Finally, HDAC3 inhibition increased PD-L1 expressionon dendritic cells in the tumor microenvironment.Combining selective HDAC3 inhibitor with anti–PD-L1immunotherapy enhanced tumor regression in syngeneicmurine lymphoma model. Our findings identify HDAC3 asan important epigenetic regulator of PD-L1 expressionand implicate combination of HDAC3 inhibition withPD-1/PD-L1 blockade in the treatment of B-cell lymphomas.

IntroductionThe programmed cell-death protein 1 (PD-1)/programmed

cell death-ligand 1 (PD-L1) axis has been characterized as apotent inhibitor of immune activation and an important mech-anism underlying tumor immune escape (1, 2). PD-1 is gen-erally highly expressed on tumor-infiltrating T cells. Interactionof PD-1 with its ligand PD-L1 on tumor cells or immune cellscan inhibit the initial activation of T cells and suppress effectorT-cell generation and functions, including cytokine productionand cytotoxicity (3, 4). PD-L1 is expressed in various types ofhuman cancer including Hodgkin lymphoma (5) and diffuselarge B-cell lymphomas (DLBCL; ref. 6), and suppresses anti-tumor T-cell response (7). Blockade of the PD-L1/PD-1 axis canreactivate antitumor immunity and induce durable clinicalresponse against a growing list of human cancers includingB-cell lymphoma (1, 2, 8). In cancer patients, the clinicalresponse to PD-L1/PD-1 immunotherapy correlates with tumor

and host PD-L1 expression, along with other predictive bio-markers such as tumor-infiltrating CD8þ T cells and mutationburden (9, 10). Accumulating evidences have indicated thataltered PD-L1 expression by small molecules can modify theefficacy of anti–PD-1/PD-L1 therapy in preclinical mouse mod-el. For example, CDK4/6 inhibitor synergizes with anti–PD-1antibody to elicit an enhanced therapeutic efficacy by increas-ing tumoral PD-L1 expression (11).

PD-L1 expression on tumor cells can be regulated at bothtranscriptional and posttranslational levels (7). The transcrip-tional control of PD-L1 has been extensively studied duringthe past several years. Many transcription factors, includingIRF1, STAT1/3, and MYC, have been reported to bind to thepromoter region of PD-L1 gene and regulate its transcrip-tion (12–15). Inflammatory signals modulate PD-L1 geneexpression via these transcription factors. For example,IFNg , a potent inducer of PD-L1 transcription, functions byactivating the JAK/STAT/IRF1 pathway (13). These transcriptionfactors directly bind to the transcriptional regulatory elements ofPD-L1 gene and alter chromatin status via their associated epige-netic regulators. Histone deacetylases (HDAC) are ubiquitouslyexpressed epigenetic regulators by removing acetyl groups fromtheN-acetyl lysine amino acid on the tail of histone to silence genetranscription (16). HDACs have been found to deacetylate non-histone proteins that are associatedwith various functions such asgene expression (17). Various HDAC isoforms differ in theirsubcellular location and targets, which account for their differentbiological functions. Recently, pan-HDACs or class-specificHDAC inhibitors have been reported to induce PD-L1 expressionin lung cancers and melanoma (18, 19), suggesting that HDACsare involved in the regulation of PD-L1. However, which HDACisoform is crucial for the regulation of PD-L1 and the molecularmechanisms of action remain unknown.

Shanghai Institute of Immunology and Department of Immunology and Micro-biology, Key Laboratory ofCell Differentiation andApoptosis of ChineseMinistryof Education, Shanghai Jiao Tong University School of Medicine, Shanghai,China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

S. Deng and Q. Hu contributed equally to this article.

Corresponding Authors: Chuanxin Huang, Phone: 86-21-54610161; E-mail:[email protected]; and Cheng Peng, Phone: 86-21-54616725; E-mail:[email protected]. Shanghai Institute of Immunology, Shanghai Jiao TongUniversity School of Medicine, Shanghai 200032, China.

doi: 10.1158/1535-7163.MCT-18-1068

�2019 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 18(5) May 2019900

on June 3, 2020. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 1, 2019; DOI: 10.1158/1535-7163.MCT-18-1068

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-18-1068&domain=pdf&date_stamp=2019-4-18

http://mct.aacrjournals.org/

HDAC inhibitors are well known to affect cancer cell viabilityand are already in use for the treatment of some subtypes ofhematologic malignancies (20, 21). In addition to direct cyto-toxicity, HDAC inhibitors can alter the immune landscape oftumor cells, through changes in expression of costimulatoryand coinhibitory molecules, MHC and tumor antigens, aswell as cytokine production by tumor cells (21–23). HDACinhibitors can also affect the phenotypes of different immunecell subsets in the tumor environment and draining lymphnode (24, 25). HDAC inhibitors have been proposed to poten-tially synergize with immunotherapies (21, 26). NonselectiveHDAC inhibitors have been recently reported to augment theantitumor effect of anti–PD-1 antibody by increasing PD-L1expression in syngeneic murine tumor models including lungcancers and melanoma (18, 27). We reasoned that HDACinhibition may be a rational therapeutic strategy to be imple-mented in combination with PD-1 blockade for the treatmentof B-cell lymphoma. However, nonselective HDAC inhibitorstarget multiple HDACs and may cause serious unfavorabletoxicities such as thrombocytopenia, fatigue, and diarrhea,limiting their clinical application (28). It is thereby ideal tofind out HDAC isoform–specific inhibitors which can be usedto promote PD-L1 expression and the clinical response to PD-1immunotherapy while avoiding the adverse events associatedwith pan-HDAC inhibition.

In this study, we report that, in B-cell lymphomas, HDAC3 is akey regulator of PD-L1 transcription via direct and indirectmechanisms, and HDAC3 inhibition augments the response toanti–PD-1 blockade in a syngeneic murine lymphoma model.

Materials and MethodsReagents

NonselectiveHDAC inhibitors SAHA, LBH589 (panobinostat),valproic acid sodium salt (VPA), trichostatin A (TSA), andHDAC1/2-slective inhibitor romidepsin as well as HDAC3-selective inhibitor RGFP966 were purchased from SelleckChemicals, and its chemical structure was described previous-ly (29). Bromodomain inhibitor JQ1 and DNAmethyltransferaseinhibitor 5-Azacitidine (5-Aza) were also obtained from SelleckChemicals. The BCL6 BTB inhibitor FX1 was provided by profes-sor Ari Melnick (Weill Cornell Medical College).

Cell line culture, siRNA electroporation, and PD-L1 knockoutcells

The B-lymphoma cells lines A20, OCI-LY1, and TMD8 wereobtained from the ATCC and authenticated via short tandemrepeat profiling. OCI-LY1 cells were maintained in mediumcontaining 90% Iscove and 10% FCS and supplementedwith antibiotics. A20 and TMD8 cells were maintained inmedium containing 90% RPMI and 10% FCS supplementedwith antibiotics, L-glutamine, and 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) Potential contamina-tion by mycoplasma was often checked using the LookoutMycoplasma PCR Detection Kit purchased from Sigma-Aldrich.Scramble siRNA, HDAC3 siRNA (#1: 5-CCCGGTGTTGGACA-TATGAAA-3 and #2: 5-GACACCCAATGAAACCTCATCGCCT-3), and BCL6 siRNA (5-CCATTGTGAGAAGTGTAACCTGCAT-3) were purchased from Shanghai GenePharma Company.siRNAs were transiently electroporated into lymphoma celllines using cell line nucleofector transfection kit (AMAXA)

according to the manufacturer's instruction. To generate PD-L1 knockout (PD-L1ko) cells, A20 cells were transiently trans-fected with a Cas9-single guide RNA (sgRNA) expression vector(pSpCas9(BB), Addgene) targeting PD-L1 or control guideRNAs. PD-L1 sgRNA, 50-GTATGGCAGCAACGTCACGA-30. Tendays after transfection, PD-L1ko cells were sorting on PD-L1–negative cells by flow cytometer.

mRNA extraction and quantitative RT-PCRTotal RNA was prepared with Trizol reagent (Invitrogen), and

cDNAwas synthesized using Superscript reverse transcriptase andrandomprimers (Invitrogen).Quantitative real-time PCR (qPCR)was performed using the Power SYBR Green PCR master mix(Vazyme) on an ABI Prism sequence detection system (AppliedBiosystems). Gene-specific primers are listed in SupplementaryTable S1.

Flow cytometry and antibodiesSingle-cell suspensions were prepared from freshmouse tumor

tissues or cultured cells. Antibodies used for staining were listedin the Supplementary Data. Cell surface staining was performedin FACS buffer (PBS with 2% FBS, 2 mmol/L EDTA, and 0.05%sodium azide). All flow cytometry data were acquired on aFortessa X-20 (BD Biosciences), and live cells were gated foranalysis with FlowJo software (Tree Star).

In vivo mouse modelSix- to 8-week-old wild-type BALB/c mice were obtained

from the Shanghai SLAC Laboratory Animal Co. Ltd. Mice weremaintained in a specific pathogen-free facility, and all animalexperiments were performed in accordance to protocols approvedby the Institutional Animal Care and Use Committee ofShanghai Jiao Tong University, School of Medicine. Mice weres.c. inoculated with 5 � 106A20 cells. RGFP966 was injected bythe i.p. route at a dose of 50mg/kg beginning on day 7 after tumorimplantation and continued daily for 21 days. Alternatively,200 mg anti–PD-L1 blocking antibodies (BE101) from BioXCellwere injected by i.p. for 3 times (once every 3 days) from day 7after tumor implantation. Solvent (2%DMSO, 30%PEG300, and5% Tween 80) was used in the treatment control group. Tumorsize were monitored with a digital caliper every 3 days andexpressed as volume (0.5 � length � width � height).

Statistical analysisAnalyses were performed using Prism 6.0 (GraphPad). Statis-

tical significance was calculated using the two-tailed, unpairedStudent t test or one-way ANOVA, as specified. P values < 0.05were considered statistically significant.

Other Materials and Methods are provided as SupplementaryData.

ResultsPan-HDAC inhibitors upregulate expression of PD-L1 inmurine and human B-cell lymphoma cells

Pan- and selective class I/IV HDAC inhibitors have beenreported to increase PD-L1 expression in melanoma and lungcancer cell lines (18, 19). We hypothesized that HDAC inhibitioncould also upregulate PD-L1 expression in B-cell lymphoma. A20lymphoma cells were treated with SAHA, a pan HDAC inhibitor,for 48hours at doses ranging from0.1 to 1mmol/L that caused less

HDAC3 as a Critical Negative Regulator of PD-L1

www.aacrjournals.org Mol Cancer Ther; 18(5) May 2019 901




than 30% cell death at 48 hours (Supplementary Fig. S1A).SAHA treatment significantly increased surface PD-L1 levels ina dose-dependent manner (Fig. 1A). Note that 1 mmol/L SAHAinduced an approximately 3-fold increase in PD-L1 levels. Theclinically achievable concentration of SAHA is 500 nmol/L to4 mmol/L (30). Accordingly, we used 1 mmol/L SAHA for the

following experiments. Of note, upregulation of surface PD-L1levels started as early as 12 hours after SAHA treatment withoutobvious cell death (Fig. 1B; Supplementary Fig. S1B). Consistentwith previous reports (31, 32), 1 mmol/L SAHA upregulatedthe expression of many costimulatory molecules such as CD80,CD86,MHC I, andMHC II in A20 cells (Supplementary Fig. S1C).

Figure 1.

Pan-HDAC inhibitor SAHA promotesPD-L1 expression in B-cell lymphomacells. A, Flow cytometry of surfacelevels of PD-L1 on A20 cells treatedwith different doses of SAHA for 48hours. B, Flow cytometry of surfacelevels of PD-L1 on A20 cells treatedwith 1 mmol/L SAHA for indicatedtimes. C, The relative mRNAexpression levels of PD-L1 in A20 cellstreated with 1 mmol/L SAHA forindicated times, determined by RT-qPCR.D, Flow cytometry of surfacelevels of PD-L1 on OCI-LY1 and TMD8cells treated with 1 mmol/L SAHA for48 hours. Right plot, quantification ofrelative mean fluorescence intensity(MFI) of PD-L1. Data are mean� SD ofthree independent experiments. �� ,P < 0.01 (two-tailed unpaired t test).

Figure 2.

SAHA treatment increases histone acetylation and BRD4 recruitment at the PD-L1 promoter.A and B, Histone acetylation and Brd4 occupation at the PD-L1promoter. A20 cells were treated with 1 mmol/L SAHA for 9 hours before being subjected to ChIP assay using indicated anti–acetyl-histone antibodies oranti-BRD4 antibodies followed by qPCR analysis using primers targeting indicated PD-L1 promoter regions. C, Flow cytometry of surface levels of PD-L1 onA20 cells treated with indicated inhibitors for 48 hours. Right, quantification of relative MFI of PD-L1. D, Flow cytometry of surface levels of PD-L1 on OCI-LY1and TMD8 cells treated with indicated inhibitors for 48 hours. Right, quantification of relative MFI of PD-L1. Data are mean� SD of three independentexperiments. ns, not significant; �� , P < 0.01 (two-tailed unpaired t test).

Deng et al.

Mol Cancer Ther; 18(5) May 2019 Molecular Cancer Therapeutics902




Real-time PCR assay further showed that SAHA treatmentincreased the mRNA abundance of PD-L1, indicating that SAHAregulates PD-L1 expression at the transcriptional levels (Fig. 1C).Upregulation of surface expression of PD-L1 and its transcript wasobserved in two human DLBCL cell lines OCI-LY1 and TMD8treated with SAHA (Fig. 1D; Supplementary Fig. S1D). Finally,both TSA and LBH589, two pan-HDAC inhibitors, also inducedPD-L1 expression in A20 cells (Supplementary Fig. S1E). Collec-tively, these results demonstrate that HDAC inhibition canincrease the transcription and surface expression of PD-L1 inB-cell lymphomas.

Pan-HDAC inhibitor increases histone acetylation at the PD-L1promoter and promotes PD-L1 transcription via BRD4recruitment

We next sought to understand howHDAC inhibition increasesPD-L1 transcription in B-cell lymphoma cells. HDAC inhibitorsare known to modulate global gene expression mainly via thealteration of acetylation "marks" on histones, which opens up

chromatin structures and recruits "acetyllysine reader" to theacetylated sites, subsequently activating downstream target geneexpression (16). HDAC inhibition has been reported tomodulategene transcription by enhancing acetylation of histone H3 atlysine 4 (H3K4ac), lysine 9 (H3K9ac), or lysine 27 (H3K27ac)of promoters. Accordingly, we performed chromatin immuno-precipitation (ChIP) assay using antibodies against H3K4ac,H3K9ac, and H3K27ac in A20 cells treated with SAHA for 9 hoursfollowed by qPCR analysis using three different pairs of primers(Fig. 2A). SAHA treatment increased the acetylation of H3K4,H3K9, and H3K27 at the promoter region of PD-L1 gene, withinan approximately 1.5 kb region 50 to its transcription start site(Fig. 2A).

According to previous reports, the bromodomain and extra-terminal protein BRD4 directly binds to acetylated lysine onhistone tails to facilitate gene transcription by RNA polymeraseII (33). IFNg treatment enhanced the H3K27ac mark and BRD4recruitment at the promoter region of PD-L1 in melanomacells (34, 35). In addition, BRD4 inhibition or knockdown

Figure 3.

HDAC3 is a crucial negative regulator of PD-L1 in B-cell lymphoma. A, Immunoblotting of indicated histone acetylation marks in A20 transfected withdifferent doses of indicated inhibitors for 9 hours. B, Surface levels of PD-L1 on A20, OCI-LY1, or TMD8 cells treated with indicated inhibitors for 48 hours,determined by flow cytometry. C, The relative mRNA expression levels of PD-L1 in A20, OCI-LY1, and TMD8 cells treated with 5 mmol/L RGFP966 for 24 hours.D, Immunoblotting of HDAC3 protein in scramble (scr) or HDAC3 siRNA-transfected A20 cells for 3 days. E, Flow cytometry analysis of surface levels of PD-L1 onHDAC3-knockdown A20 cells and quantification. Data are mean� SD of three independent experiments. � , P < 0.01 (two-tailed unpaired t test). F, Correlationbetween HDAC3 and PD-L1 was determined by Spearman statistical analysis in 203 cases of DLBCLs.






inhibited basal or IFNg-induced PD-L1 transcription (13).We, therefore, tested whether BRD4 was involved in HDACinhibition–mediated PD-L1 transcription. ChIP-qPCR assayshowed that SAHA treatment increased BRD4 occupancy atthe promoter region of PD-L1 gene in A20 cells (Fig. 2B).Furthermore, JQ1, a pharmacologic inhibitor of BRD4 that func-tions by dissociating BRD4 from the chromatin (33), dramaticallyblocked SAHA-induced PD-L1 upregulation in A20 cells (Fig. 2C;Supplementary Fig. S2), whereas JQ1 alone had only a marginaleffect on basal PD-L1 expression (Supplementary Fig. S2). JQ1treatment could also prevent SAHA-induced PD-L1 upregulationin OCI-LY1 and TMD8 cells (Fig. 2D). Collectively, these resultsdemonstrate that HDAC inhibition upregulates PD-L1 transcrip-tion by increasing histone acetylation of the PD-L1 promoter.

HDAC3 inhibition promotes PD-L1 expression in B-celllymphomas

We next investigated which HDAC isoform plays an importantrole in modulating PD-L1 transcription. We focused particularlyon Class I HDACs because they are ubiquitously expressed byB-cell lymphomas and regulate gene transcription mainly byaffecting the acetylation status of histone tails. Class I HDACshave four members including HDAC1, HDAC2, HDAC3, andHDAC8. HDAC1, HDAC2, and HDAC3 are well characterizedandpotential therapeutic targets for B-cell lymphomas. BothClassI HDAC inhibitor VPA and selective HDAC1/2 inhibitor Romi-depsin dramatically increased acetylated histone H3 (H3ac) andacetylation ofH3K27 in A20 cells 9 hours after treatment, whereas

selective HDAC3 inhibitor RGFP966 resulted in a less profound,but observable increase in H3ac and H3K27ac (Fig. 3A). More-over, VPA, romidepsin, or RGFP966 impaired cell survivaland upregulated the surface levels of many costimulatory mole-cules such as CD80, CD86, and MHC I in A20 cells although to adifferent extent at 2 days of treatment (Supplementary Fig. S3Aand S3B). Interestingly, treatment with either VPA or RGFP966markedly enhanced surface PD-L1 levels in A20 cells and twohuman B-cell lymphoma cells at 2 days of treatment (Fig. 3B). Incontrast, Romidepsin had no appreciable or weak effect onexpression of PD-L1 in these cell lines (Fig. 3B). Similar to SAHA,RGFP966 treatment also increasedmRNA abundance of PD-L1 inthese cell lines (Fig. 3C). Moreover, siRNA-mediated depletion ofHDAC3upregulated surface PD-L1 levels inA20 cells (Fig. 3D andE). Finally, the mRNA expression levels of HDAC3 and PD-L1were inversely correlated in 203 cases of human DLBCLs(Fig. 3F).These results indicate that HDAC3 is a critical HDAC isoform thatinhibits PD-L1 expression in B-cell lymphoma.

HDAC3 is recruited to the PD-L1 promoter by BCL6 to inhibithistone acetylation at the PD-L1 promoter

Our aforementioned results showed that pan-HDAC inhibitorspromoted PD-L1 transcription by increasing histone acetylationat the PD-L1 promoter (Fig. 2). We next investigated whetherHDAC3 inhibition upregulated PD-L1 expression via thesimilar mechanism. ChIP-qPCR assay showed that RGFP966treatment increased acetylation of H3K27 at the promoter regionof PD-L1 (Fig. 4A). Accordingly, JQ1 treatment largely reversed

Figure 4.

HDAC3 and SMRT corepressors are recruited to the PD-L1 promoter by BCL6. A, ChIP-qPCR analysis of H3K27ac at the PD-L1 promoter in A20 cells treated with5 mmol/L RGFP966 for 9 hours. B, Flow cytometry of surface levels of PD-L1 on A20 cells treated with indicated inhibitors for 48 hours. Right, quantification ofrelative MFI. C, ChIP-seq tracks of BCL6 and SMRT at the PD-L1 gene locus in OCI-LY1 cells (left). ChIP-qPCR analysis of enrichment of HDAC3 and SMRT at thePD-L1 promoter in OCI-LY1 cells (right). D, HDAC3 was precipitated using anti-BCL6 antibodies in A20 cells. E, ChIP-qPCR analysis of HDAC3 enrichment at thePD-L1 promoter and immunoblotting of BCL6 protein in BCL6-knockdown OCI-LY1 cells. F, Flow cytometry of surface levels of PD-L1 on A20 cells treated with25 mmol/L FX1 for 48 hours and quantification. Data are mean� SD of three independent experiments. ns, not significant; � , P < 0.05; and �� , P < 0.01 (two-tailedunpaired t test).

Deng et al.





RGFP966-enabled PD-L1 upregulation in A20 cells (Fig. 4B).These results indicate that HDAC3 inhibition induces histoneacetylation to facilitate PD-L1 transcription.

We next sought to identify the mechanism by which HDAC3regulates histone acetylation of the PD-L1 promoter. HDAC3lacks DNA-binding domain and is unable to directly bind toDNA. HDAC3 is frequently associated with SMRT and NCORcorepressor complex that are recruited to the regulatory sequencesof target genes by transcription factors (36). The transcriptionrepressor BCL6 (B-Cell Lymphoma 6) is a master oncogene inB-cell lymphoma and modulates gene transcription throughrecruitment of SMRT/NCOR/HDAC3 repressor complex (37–39).Previous ChIP-seq studies demonstrated that BCL6 directlybinds to the promoter of PD-L1 and is suggested to regulate itstranscription. By analyzing our published ChIP-seq of humangerminal center (GC)-derived lymphoma cell line OCI-LY1 (GEOnumber: GSE29282; ref. 39), we found that BCL6 and SMRTcobound to the promoter of PD-L1 gene in OCI-LY1 (Fig. 4C).ChIP-qPCR assay confirmed that HDAC3, SMRT, and BCL6bound to the same locus at the promoter region of PD-L1 inthese cells (Fig. 4C). HDAC3 was coimmunoprecipitated withanti-BCL6 antibodies, but not control IgG in A20 cells (Fig. 4D).Moreover, siRNA-mediated BCL6 knockdown significantlyreduced the enrichment of HDAC3 at the PD-L1 promoter(Fig. 4E). Finally, BCL6 BTB inhibitor FX1, which disrupts theinteraction between the BCL6 BTB domain and SMRT/HDAC3complex, increased PD-L1 expression (Fig. 4F). Collectively, theseresults suggest a model in which HDAC3–SMRT corepressorcomplex was recruited to the promoter of PD-L1 by BCL6 tomodulate histone acetylation.

HDAC3 inhibition promotes PD-L1 transcription in part byreducing DNMT1 protein

DNA methyltransferase 1 (DNMT1) is required to maintainDNA methylation across the genome (40). 5-Aza mainly targets

DNMT1 and DNMT1 knockdown upregulates hypermethylatedendogenous retrovirus genes in cancer cells, subsequently causingan IFN response to activate immune-related genes includingPD-L1 (41, 42). Interestingly, these 5-Aza–induced genes are notgenerallymethylated at promoter regions (41, 42). Recently, it hasbeen uncovered that HDAC3 inhibition led to degradation ofDNMT1 in multiple myeloma (43). We reasoned that HDAC3inhibition might reduce DNMT1 protein level, subsequentlyactivating PD-L1 transcription in A20 cells. We observed thattreatment with either SAHA or RGFP966 resulted in a decreaseof DNMT1 protein abundance in a dose-dependent manner(Fig. 5A). Interestingly, DNMT1 protein levels did not change12hours after treatment (Fig. 5A), andPD-L1 started to increase asearly as 12 hours (Fig. 5B), suggesting that DNMT1 degradationcontributes to PD-L1 upregulation at late-time point. Consistentwith this, 5-Aza increased PD-L1 expression 48 hours after treat-ment (Fig. 5B). Interestingly, the PD-L1 promoter was hypo-methylated in A20 cells, and RGFP966 treatment did not causenoticeable changes in DNA methylation patterns in the PD-L1promoter (Supplementary Fig. S4). Notably, simultaneous treat-ment with 5-Aza and RGFP966 caused a more striking upregula-tion of surface PD-L1 in A20 cells as compared with treatmentwith each single inhibitor (Fig. 5B). Taken together, these resultsindicate that HDAC3 inhibition induces DNMT1 degradation,subsequently activating PD-L1 transcription.

RGFP966 increases PD-L1 levels on both tumor cells anddendritic cells in the tumor microenvironment in syngeneicmurine lymphoma model

We next asked whether HDAC3 inhibition increases PD-L1expression on tumor cells in syngeneicmurine lymphomamodel.To this end,micewere injected s.c. withA20 cells. Seven days later,mice were administrated with RGFP966 (50 mg/kg) for 9 conse-cutive days, and tumor tissues were then removed for flow cyto-metric analysis of PD-L1 expression. This dose of RGFP966 has

Figure 5.

HDAC3 inhibition reduces DNMT1 proteinlevel. A, Immunoblotting of DNMT1protein in A20 cells treated with SAHA orRGFP966. B, Flow cytometry of surfacelevels of PD-L1 on A20 cells treated withindicated inhibitors for 12, 24, or 48 hours.Data are mean� SD of three independentexperiments. � , P < 0.01 and �� , P < 0.01(two-tailed unpaired t test).






been previously described (29). We observed that RGFP966treatment led to a 2-fold increase in PD-L1 expression ontumor cells (Supplementary Fig. S5A). Because PD-L1 expressionon host cells also affects the antitumor activity of anti–PD-L1immunotherapy (44, 45), we investigated the effect of RGFP966on PD-L1 expression on immune cells including dendritic cells(DC, CD11cþ), myeloid-derived suppression cells (MDSC,CD11bþGr1þ), andmacrophages (CD11bþF4/80þ) in the tumormicroenvironment and draining lymphoid nodes. An increase inthe expression of PD-L1 was observed on tumor-infiltrating DCs,but not MDSCs and macrophages (Supplementary Fig. S5B).PD-L1 expression was not significantly altered in these immunecells in the draining lymphoid nodes (Supplementary Fig. S5C).Taken together, HDAC3 inhibition increased PD-L1 expressionon tumor cells and on DCs in the tumor environment in vivo.

RGFP966 enhanced the therapeutic effect of anti–PD-L1blockade in syngeneic murine lymphoma model

Recent preclinical studies have revealed that both tumor andhost-derived PD-L1 can suppress antitumor immunity (44–46).To clarify the relative contributions of PD-L1 upregulation ontumor cells in inhibiting the therapeutic response to HDAC3inhibition, we generated PD-L1KO A20 cells (SupplementaryFig. S6A). PD-L1 deficiency did not affect cell growth in vitro, butsignificantly delayed tumor growth (Supplementary Fig. S6B and

S6C). Moreover, RGFP966 treatment retarded PD-L1KO, but notwild-type tumor growth, indicating that RGFP966-induced upre-gulation of PD-L1 represents an important mechanism underly-ing RGFP966 resistance. We next investigate whether HDAC3inhibition might enhance the antitumor effect of anti–PD-L1therapy in vivo. To test this hypothesis, A20 cells were implanted,and 7 days later, tumors were treated with RGFP966 (50 mg/kg),anti–PD-L1 alone (200 mg per mouse), or a combination of bothagents (Fig. 6A). Treatment with RGFP966 alone did not affecttumor growth and failed to lead to tumor regression (0/6). Anti–PD-L1 antibodies alone effectively controlled tumor growth, and2 of 10 tumors were finally cleared. Interestingly, RGFP966 incombinationwith anti–PD-L1 antibodymarkedly retarded tumorprogression and resulted in 7 complete responses out of the 11treated mice (Fig. 6B). As expected, SAHA treatment couldincrease the antitumor effect of anti–PD-L1 antibody in syngeneicmurine lymphoma model (Supplementary Fig. S7). These resultsdemonstrate that HDAC3 inhibitor synergized with anti–PD-L1therapy to elicit an enhanced therapeutic efficacy.

DiscussionThe epigenetic regulation of PD-L1 in B-cell lymphoma

remains to be elucidated. Here, we found that HDAC inhibitorscan increase themRNA abundance and surface levels of PD-L1 on

Figure 6.

Combinatorial therapy with anti–PD-L1 antibodies and RGFP966 in syngeneic B-cell lymphomamodel. BALB/c mice were inoculated s.c. with A20 lymphomacells. Seven days after inoculation, mice began to receive RGFP966 (50mg/kg), anti–PD-L1 blocking antibodies (200 mg), a combination of the two reagents,or solvent control as indicated (A). Tumor volumes of mice treated with control (n¼ 6), RGFP966 (n¼ 6), anti–PD-L1 antibodies (n¼ 10), or combined therapy(n¼ 11) were measured every 3 days and plotted individually (B). Data are from two independent experiments. C,Aworking model of how HDAC3 inhibitionupregulates PD-L1 transcription. The SMRT–HDAC3 corepressor complex is recruited by BCL6 to the PD-L1 promoter. HDAC3 inhibition induces a rapid increasein histone acetylation of the PD-L1 promoter and subsequent recruitment of BRD4, promoting its transcription. This process can be blocked by JQ1. In addition,HDAC3 inhibition induces DNMT1 degradation to upregulate PD-L1 expression potentially by triggering an IFN response. The increase in PD-L1 may represent asevere clinical problem for patients receiving HDAC3 inhibitor treatment and may be one of the underlying mechanisms accounting for HDAC3 inhibitorresistance via evasion of immune surveillance checkpoints. Hence, our study provides a molecular mechanism as well as the rationale for the combinatorialtreatment of PD-1/PD-L1 blocking and selective HDAC3 inhibitor as a more efficient anticancer option in clinic.

Deng et al.





a panel of murine and human B-cell lymphoma cell lines (Fig. 1).Mechanistically, HDAC inhibition led to a rapid increase inhistone acetylation at the promoter region of PD-L1 and subse-quent recruitment of BRD4 to drive its transcription (Fig. 2).Accordingly, BRD4 inhibitor JQ1 dramatically suppressed theupregulation of PD-L1 triggered by HDAC inhibition.

Using specific HDAC inhibitors and gene knockdown, wefound that HDAC3, but not HDAC1/2, is the key HDACisoform that is responsible for the regulation of PD-L1 tran-scription (Fig. 3). HDAC3 is likely to be the primary enzymeresponsible for the deacetylase activity that is associated withSMRT-mediated repressive events. ChIP-seq further showedthat BCL6 recruited HDAC3–SMRT corepressor complex to thePD-L1 promoter (Fig. 4). Therefore, BCL6–HDAC3–SMRT com-plex appears to locate to the PD-L1 promoter to suppress itstranscription by directly deacetylating histones (Fig. 6C). Inaddition to BCL6, other transcription factors may be involvedin the recruitment of SMRT–HDAC3 complex to the PD-L1promoter. In addition to increased histone acetylation at thePD-L1 promoter, DNMT1 degradation may contribute to PD-L1upregulation at late time point after HDAC3 inhibition(Fig. 6C). HDAC3 inhibition did not directly affect DNAmethylation of the PD-L1 promoter (Supplementary Fig. S4),but may trigger an IFN response to activate PD-L1 expressionvia DNMT1 degradation.

Many types of B-cell lymphomas depend on HDAC3 fortheir survival and proliferation (47, 48). Targeting HDAC3 hasbeen apromising therapy for B-cell lymphoma.However,HDAC3inhibition–induced increase of PD-L1 could be one of the under-lying mechanisms accounting for HDAC3 inhibitor resistance viaevasion of immune surveillance checkpoints (Fig. 6C). Thisnotion is supported by our observation that RGFP966 signifi-cantly retarded PD-L1–deficient, but not wild-type, tumor growth(Supplementary Fig. S6). RGFP966 failed to eradicate PD-L1–deficient lymphomas. In contrast, combination of RGFP966 andanti–PD-L1 antibody can completely eradicate the majority ofwild-type lymphoma (Fig. 6B). Therefore, RGFP966 augments thetherapeutic effect of PD-L1 blockade by increasing PD-L1 expres-sion on tumor and DCs. Of note, HDAC3 is ubiquitouslyexpressed in tumor cells and effector immune cells within tumormicroenvironment. HDAC inhibitors have been reported toelevate T-cell chemokine expression to argument responses

to PD-1 immunotherapy in lung cancer (49). In addition toPD-L1, HDAC3 inhibition may modulate immune-relatedgenes in these cells to enhance anti–PD-L1 therapy. Therefore,PD-L1 upregulation represents an important, but not unique,mechanism by which RGFP966 augments the response to anti–PD-L1 therapy.

In summary, we identified HDAC3 as an important epigeneticregulator of PD-L1 in B-cell lymphoma and the molecularmechanisms by which HDAC3 suppresses PD-L1 transcription.Selective HDAC3 inhibitor facilitates the clinical response toPD-L1 immunotherapy while avoiding adverse events associatedwith pan-HDAC inhibition. Our study also provides thepreclinical rationale for combination of selective HDAC3 inhibi-tors with PD-1/PD-L1 immunotherapy in the treatment of B-celllymphomas.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. HuangDevelopment of methodology: S. Deng, Q. Hu, C. Peng, C. HuangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Deng, Q. Hu, H. Zhang, F. Yang, C. PengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Deng, Q. Hu, H. Zhang, F. Yang, C. Peng, C. HuangWriting, review, and/or revision of the manuscript: C. Peng, C. HuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Peng, C. HuangStudy supervision: C. Peng, C. Huang

AcknowledgmentsWe thank professor Ari Mlenick (Weill Cornell Medical College) for

providing FX1. This work was supported by the National Natural ScienceFoundation of China (grant numbers: 31870872, to C. Huang; 31800755, toC. Peng) and the 1000-Youth Elite Program.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 18, 2018; revised January 29, 2019; accepted February22, 2019; published first March 1, 2019.

References1. Zou W, Wolchok JD, Chen L. PD-L1 (B7-H1) and PD-1 pathway blockade

for cancer therapy: mechanisms, response biomarkers, and combinations.Sci Transl Med 2016;8:328rv4.

2. Chen L, Han X. Anti-PD-1/PD-L1 therapy of human cancer: past, present,and future. J Clin Invest 2015;125:3384–91.

3. Hirano F, Kaneko K, Tamura H, Dong H, Wang S, Ichikawa M, et al.Blockade of B7-H1 and PD-1 bymonoclonal antibodies potentiates cancertherapeutic immunity. Cancer Res 2005;65:1089–96.

4. Francisco LM, Salinas VH, BrownKE, Vanguri VK, FreemanGJ, KuchrooVK,et al. PD-L1 regulates the development, maintenance, and function ofinduced regulatory T cells. J Exp Med 2009;206:3015–29.

5. Green MR, Monti S, Rodig SJ, Juszczynski P, Currie T, O'Donnell E, et al.Integrative analysis reveals selective 9p24.1 amplification, increased PD-1ligand expression, and further induction via JAK2 in nodular sclerosingHodgkin lymphoma and primary mediastinal large B-cell lymphoma.Blood 2010;116:3268–77.

6. Kiyasu J, Miyoshi H, Hirata A, Arakawa F, Ichikawa A, Niino D, et al.Expression of programmed cell death ligand 1 is associated with poor

overall survival in patientswithdiffuse largeB-cell lymphoma. Blood2015;126:2193–201.

7. Sun C,Mezzadra R, Schumacher TN. Regulation and function of the PD-L1checkpoint. Immunity 2018;48:434–52.

8. Goodman A, Patel SP, Kurzrock R. PD-1-PD-L1 immune-checkpointblockade in B-cell lymphomas. Nat Rev Clin Oncol 2017;14:203–20.

9. Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, et al.Predictive correlates of response to the anti-PD-L1 antibody MPDL3280Ain cancer patients. Nature 2014;515:563–7.

10. Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al.PD-1 blockade induces responses by inhibiting adaptive immune resis-tance. Nature 2014;515:568–71.

11. Zhang J, Bu X, Wang H, Zhu Y, Geng Y, Nihira NT, et al. Cyclin D-CDK4kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immunesurveillance. Nature 2018;553:91–5.

12. Casey SC, Tong L, Li Y, Do R,Walz S, Fitzgerald KN, et al.MYC regulates theantitumor immune response throughCD47 and PD-L1. Science 2016;352:227–31.






13. Garcia-Diaz A, Shin DS, Moreno BH, Saco J, Escuin-Ordinas H, RodriguezGA, et al. Interferon receptor signaling pathways regulating PD-L1 andPD-L2 expression. Cell Rep 2017;19:1189–201.

14. Lee SJ, Jang BC, Lee SW,Yang YI, Suh SI, Park YM, et al. Interferon regulatoryfactor-1 is prerequisite to the constitutive expression and IFN-gamma-induced upregulation of B7-H1 (CD274). FEBS Lett 2006;580:755–62.

15. Atsaves V, Tsesmetzis N, Chioureas D, Kis L, Leventaki V, Drakos E, et al.PD-L1 is commonly expressed and transcriptionally regulated by STAT3andMYC inALK-negative anaplastic large-cell lymphoma. Leukemia 2017;31:1633–7.

16. Scholz C, Weinert BT, Wagner SA, Beli P, Miyake Y, Qi J, et al. Acetylationsite specificities of lysine deacetylase inhibitors in human cells.Nat Biotechnol 2015;33:415–23.

17. Halsall JA, Turner BM. Histone deacetylase inhibitors for cancer therapy:an evolutionarily ancient resistance response may explain their limitedsuccess. Bioessays 2016;38:1102–10.

18. Woods DM, Sodre AL, Villagra A, Sarnaik A, Sotomayor EM, Weber J.HDAC inhibition upregulates PD-1 ligands in melanoma and augmentsimmunotherapy with PD-1 blockade. Cancer Immunol Res 2015;3:1375–85.

19. Briere D, Sudhakar N, Woods DM, Hallin J, Engstrom LD, Aranda R, et al.The class I/IV HDAC inhibitor mocetinostat increases tumor antigenpresentation, decreases immune suppressive cell types and augmentscheckpoint inhibitor therapy. Cancer Immunol Immunother 2018;67:381–92.

20. Mercurio C, Minucci S, Pelicci PG. Histone deacetylases and epigenetictherapies of hematological malignancies. Pharmacol Res 2010;62:18–34.

21. West AC, Johnstone RW. New and emerging HDAC inhibitors forcancer treatment. J Clin Invest 2014;124:30–9.

22. Newbold A, Matthews GM, Bots M, Cluse LA, Clarke CJ, Banks KM, et al.Molecular and biologic analysis of histone deacetylase inhibitors withdiverse specificities. Mol Cancer Ther 2013;12:2709–21.

23. Newbold A, Falkenberg KJ, Prince HM, Johnstone RW.How do tumor cellsrespond to HDAC inhibition? FEBS J 2016;283:4032–46.

24. Akimova T, Beier UH, Liu Y, Wang L, Hancock WW. Histone/proteindeacetylases and T-cell immune responses. Blood 2012;119:2443–51.

25. Shen L, Orillion A, Pili R. Histone deacetylase inhibitors as immunomo-dulators in cancer therapeutics. Epigenomics 2016;8:415–28.

26. Hughes PE, Caenepeel S, Wu LC. Targeted therapy and checkpoint immu-notherapy combinations for the treatment of cancer. Trends Immunol2016;37:462–76.

27. Orillion A, Hashimoto A, Damayanti N, Shen L, Adelaiye-Ogala R, Arisa S,et al. Entinostat neutralizesmyeloid-derived suppressor cells and enhancesthe antitumor effect of PD-1 inhibition inmurinemodels of lung and renalcell carcinoma. Clin Cancer Res 2017;23:5187–201.

28. Gryder BE, Sodji QH, Oyelere AK. Targeted cancer therapy: giving histonedeacetylase inhibitors all they need to succeed. Fut Med Chem 2012;4:505–24.

29. Malvaez M, McQuown SC, Rogge GA, Astarabadi M, Jacques V, Carreiro S,et al. HDAC3-selective inhibitor enhances extinction of cocaine-seekingbehavior in a persistent manner. PNAS 2013;110:2647–52.

30. Kelly WK, O'Connor OA, Krug LM, Chiao JH, Heaney M, Curley T, et al.Phase I study of an oral histone deacetylase inhibitor, suberoylanilidehydroxamic acid, in patients with advanced cancer. J Clin Oncol 2005;23:3923–31.

31. 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.

32. Cycon KA, Mulvaney K, Rimsza LM, Persky D, Murphy SP. Histonedeacetylase inhibitors activate CIITA and MHC class II antigen expressionin diffuse large B-cell lymphoma. Immunology 2013;140:259–72.

33. Filippakopoulos P, Knapp S. Targeting bromodomains: epigenetic readersof lysine acetylation. Nat Rev Drug Discov 2014;13:337–56.

34. Hogg SJ, Vervoort SJ, Deswal S, Ott CJ, Li J, Cluse LA, et al. BET-bromodomain inhibitors engage the host immune system and regulateexpression of the immune checkpoint ligand PD-L1. Cell Rep 2017;18:2162–74.

35. Zhu H, Bengsch F, Svoronos N, Rutkowski MR, Bitler BG, Allegrezza MJ,et al. BET bromodomain inhibition promotes anti-tumor immunity bysuppressing PD-L1 Expression. Cell Rep 2016;16:2829–37.

36. Perissi V, Jepsen K, Glass CK, Rosenfeld MG. Deconstructing repression:evolving models of co-repressor action. Nat Rev Genet 2010;11:109–23.

37. Ahmad KF,Melnick A, Lax S, BouchardD, Liu J, Kiang CL, et al. Mechanismof SMRT corepressor recruitment by the BCL6 BTB domain.Mol Cell 2003;12:1551–64.

38. Huang C, Hatzi K, Melnick A. Lineage-specific functions of Bcl-6 inimmunity and inflammation are mediated by distinct biochemicalmechanisms. Nat Immunol 2013;14:380–8.

39. Hatzi K, Jiang Y, Huang C, Garrett-Bakelman F, Gearhart MD, Gianno-poulou EG, et al. A hybrid mechanism of action for BCL6 in B cells definedby formation of functionally distinct complexes at enhancers and promo-ters. Cell Rep 2013;4:578–88.

40. Cai Y, TsaiHC, YenRC,ZhangYW,KongX,WangW, et al. Critical thresholdlevels of DNA methyltransferase 1 are required to maintain DNA meth-ylation across the genome in human cancer cells. Genome Res 2017;27:533–44.

41. Li H, Chiappinelli KB, Guzzetta AA, EaswaranH, Yen RW, Vatapalli R, et al.Immune regulation by low doses of the DNA methyltransferase inhibitor5-azacitidine in common human epithelial cancers. Oncotarget 2014;5:587–98.

42. Chiappinelli KB, Strissel PL, Desrichard A, Li H, Henke C, Akman B, et al.Inhibiting DNA methylation causes an interferon response in cancer viadsRNA including endogenous retroviruses. Cell 2015;162:974–86.

43. Harada T, Ohguchi H, Grondin Y, Kikuchi S, Sagawa M, Tai YT, et al.HDAC3 regulates DNMT1 expression in multiple myeloma: therapeuticimplications. Leukemia 2017;31:2670–7.

44. LinH,Wei S, Hurt EM, GreenMD, Zhao L, Vatan L, et al. Host expression ofPD-L1 determines efficacy of PD-L1 pathway blockade-mediated tumorregression. J Clin Invest 2018;128:805–15.

45. Tang H, Liang Y, Anders RA, Taube JM, Qiu X, Mulgaonkar A, et al. PD-L1on host cells is essential for PD-L1 blockade-mediated tumor regression.J Clin Invest 2018;128:580–8.

46. Juneja VR,McGuire KA,Manguso RT, LaFleurMW,CollinsN,HainingWN,et al. PD-L1 on tumor cells is sufficient for immune evasion in immuno-genic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med 2017;214:895–904.

47. Jiang Y, Ortega-Molina A, Geng H, Ying HY, Hatzi K, Parsa S, et al. CREBBPinactivation promotes the development of HDAC3-dependent lympho-mas. Cancer Discov 2017;7:38–53.

48. Matthews GM, Mehdipour P, Cluse LA, Falkenberg KJ, Wang E, Roth M,et al. Functional-genetic dissection of HDAC dependencies in mouselymphoid and myeloid malignancies. Blood 2015;126:2392–403.

49. Zheng H, Zhao W, Yan C, Watson CC, Massengill M, Xie M, et al. HDACinhibitors enhance T-cell chemokine expression and augment response toPD-1 immunotherapy in lung adenocarcinoma. Clin Cancer Res 2016;22:4119–32.


Deng et al.




2019;18:900-908. Published OnlineFirst March 1, 2019.Mol Cancer Ther Siyu Deng, Qianwen Hu, Heng Zhang, et al.

PD-L1 Therapy−Lymphomas and Augments the Efficacy of Anti HDAC3 Inhibition Upregulates PD-L1 Expression in B-Cell

Updated version

10.1158/1535-7163.MCT-18-1068doi:

Access the most recent version of this article at:

Cited articles

http://mct.aacrjournals.org/content/18/5/900.full#ref-list-1

This article cites 49 articles, 18 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/18/5/900To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-18-1068

http://mct.aacrjournals.org/content/18/5/900.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/18/5/900


HDAC3InhibitionUpregulatesPD-L1Expressionin B-Cell ... · Small Molecule Therapeutics...

Documents

Transcript of HDAC3InhibitionUpregulatesPD-L1Expressionin B-Cell ... · Small Molecule Therapeutics...