CANCER IMMUNOLOGY RESEARCH | RESEARCH ARTICLE

Verteporfin Inhibits PD-L1 through Autophagy and theSTAT1–IRF1–TRIM28 Signaling Axis, ExertingAntitumor Efficacy A C

Jiyong Liang1, LuluWang2, ChaoWang1,3, Jianfeng Shen2, Bojin Su1, Anantha L. Marisetty4, Dexing Fang4,Cynthia Kassab4, Kang Jin Jeong1, Wei Zhao1, Yiling Lu1, Abhinav K. Jain5, Zhicheng Zhou1, Han Liang6,Shao-Cong Sun7, Changming Lu8, Zhi-Xiang Xu9, Qinghua Yu1, Shan Shao1, XiaoHua Chen1, Meng Gao1,Francois X. Claret1, Zhiyong Ding1, Jian Chen10, Pingsheng Chen11, Michelle C. Barton5, Guang Peng2,Gordon B. Mills12, and Amy B. Heimberger4

ABSTRACT◥

Programmed cell death 1 ligand 1 (PD-L1) is a key driver oftumor-mediated immune suppression, and targeting it with anti-bodies can induce therapeutic responses. Given the costs andassociated toxicity of PD-L1 blockade, alternative therapeuticstrategies are needed. Using reverse-phase protein arrays to assessdrugs in use or likely to enter trials, we performed a candidate drugscreen for inhibitors of PD-L1 expression and identified verte-porfin as a possible small-molecule inhibitor. Verteporfin sup-pressed basal and IFN-induced PD-L1 expression in vitro andin vivo through Golgi-related autophagy and disruption of the

STAT1–IRF1–TRIM28 signaling cascade, but did not affect theproinflammatory CIITA-MHC II cascade. Within the tumormicroenvironment, verteporfin inhibited PD-L1 expression,which associated with enhanced T-lymphocyte infiltration. Inhi-bition of chromatin-associated enzyme PARP1 induced PD-L1expression in high endothelial venules (HEV) in tumors and,when combined with verteporfin, enhanced therapeutic efficacy.Thus, verteporfin effectively targets PD-L1 through transcription-al and posttranslational mechanisms, representing an alternativetherapeutic strategy for targeting PD-L1.

IntroductionDiscovered first as a B7 family ligand of the programmed cell death 1

(PD1) encoded by theCD274 gene (1), PD-L1 is a checkpointmoleculethat initiates an inhibitory pathway, suppressing CTLs upon PD-1engagement. This mechanism that normally serves to prevent overactivation of immune responses is frequently coopted by cancer cells toevade immune surveillance (2). Subsequent studies have providedcompelling evidence that the PD-L1/PD1 cascade is a highly effectivetherapeutic target for immune checkpoint blockade therapy that yieldsdurable anticancer efficacy and prolongs patient survival (3).

PD-L1 is expressed in a variety of cancer types in either a consti-tutive (or intrinsic) or IFN-induced manner, and the mechanismscontrolling the expression of PD-L1 mRNA and protein have been thesubject of numerous studies. The STAT1–IRF1 axis plays a central rolein mediating IFN-induced PD-L1 transcription in both cancer andnoncancer cells (4). In addition to transcription regulation, epigeneticmechanisms, 30 UTR variations, andmiRNAs have roles in fine-tuningthe relative expression of PD-L1 in context-dependent settings (5).Other mechanisms control PD-L1 degradation, ubiquitination, autop-hagy, glycosylation, and recycling from plasma membrane and intra-cellular compartments (6, 7). In contrast to IFN-dependent PD-L1transcription, the mechanisms underlying intrinsic PD-L1 expressionin cancer are poorly understood.

Toxicity and cost are two key issues, among others, that limit the useof antibody approaches to immune checkpoints (8, 9). The complexityof the mechanisms controlling PD-L1 expression poses another majorchallenge for the development of small-molecule PD-L1 inhibitors.Targeting one of the mechanisms controlling either intrinsic or IFN-induced PD-L1 expression may not be sufficient because this pathwaycontrols the expression of >7,000 genes. Many of these genes areessential for immune responses, especially those along the CIITA-MHC cascade, which are crucial for cancer immunogenicity (10).

1SystemsBiology, TheUniversity of TexasMDAndersonCancerCenter, Houston,Texas. 2Clinical Cancer Prevention, The University of Texas MDAnderson CancerCenter, Houston, Texas. 3Department of Obstetrics & Gynecology, Obstetrics &Gynecology Hospital of Fu Dan University, Shanghai, China. 4Neurosurgery, TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas. 5Genes andDevelopment Graduate Program, Department of Epigenetics and MolecularCarcinogenesis, and Center for Cancer Epigenetics, The University of Texas MDAnderson Cancer Center, Houston, Texas. 6Bioinformatics and ComputationalBiology, The University of Texas MD Anderson Cancer Center, Houston, Texas.7Immunology, The University of Texas MD Anderson Cancer Center, Houston,Texas. 8The Institute of Oral Health, School of Dentistry, University of Alabamaat Birmingham, Birmingham, Alabama. 9Division of Hematology and Oncology,Comprehensive Cancer Center, University of Alabama at Birmingham, Birming-ham, Alabama. 10Department of General Surgery, Second Affiliated Hospital,Zhejiang University School of Medicine, Zhejiang Province, Hangzhou, China.11Department of Pathology, Medical School of Southeast University, Nanjing,Jiangsu, China. 12Cell, Developmental & Cancer Biology, Oregon Health andSciences University, Portland, Oregon.

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

J. Liang, L. Wang, and C. Wang contributed equally to this article.

Current address for J. Shen: Department of Ophthalmology, Ninth People'sHospital, Shanghai Jiao Tong University School of Medicine and Shanghai KeyLaboratory of Orbital Diseases and Ocular Oncology, Shanghai, China; andcurrent address for Z.-X. Xu, School of Life Sciences, Henan University,Kaifeng, China.

Corresponding Authors: Amy B. Heimberger, The University of Texas MD Ander-son Cancer Center, 1400 Holcombe Boulevard, Unit 442, Houston, TX 77030.Phone: 713-792-2400; Fax: 713-794-4950; E-mail: aheimber@mdanderson.org;Jiyong Liang, jyliang@mdanderson.org; and Gordon B. Mills, Oregon Health andSciences University, Portland, OR 97239. E-mail: millsg@ohsu.edu

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Thus, it is imperative to preserve the CIITA-MHC cascade andimmune responses while still targeting PD-L1 expression.

In mammalian cells, the endoplasmic reticulum (ER)-Golgi net-work plays an important role in autophagy and is a major source ofmembrane structures contributing to formation of autophago-somes (11, 12). PD-L1 is one of a large number of glycosylated proteins,including most of the MHC antigens, that are processed through theGolgi apparatus (13). The conserved oligomeric Golgi proteins (COG)in the Golgi apparatus play an important role in posttranslationalprocessing and transport of secreted peptides and proteins that areglycosylated and targeted to the plasma membranes (14). The oligo-meric Golgi complex includes 8 members (COG1-8), with the centralCOG1 surrounded by a COG2-4 forming lobe A andCOG5-8 forminglobe B (15–19). PD-L1 associates with 7 of the 8 COGs (20), whereasthe MHC molecules associate with the coatomers (20, 21), indicatingdistinct routes of processing. It is suggested that an essential role for theCOGs is maintaining posttranslational homeostasis of either proteinglycosylation or glycosylated proteins (22). However, it remainsunclear whether PD-L1 expression can be targeted selectively becausethe central mechanisms controlling PD-L1 expression, for example,IRF1-dependent transcription and posttranslational processing ofPD-L1 through the ER–Golgi network, remain insufficiently under-stood. Indeed, despite the essential role of IRF1 in PD-L1 mRNAexpression, little is known regarding how this mechanism is regulatedat the PD-L1 gene promoter and whether PD-L1 transactivation canbe dissected from other IRF1 downstream genes. As such, the goal ofthis studywas to identify potential therapeutic agents that could inhibitPD-L1 while maintaining antitumor immune responses.

Materials and MethodsCell culture

All cell lines obtained from the ATCC were curated by and redis-tributed through The University of Texas MD Anderson CancerCenter (MD Anderson, Houston, TX) Characterized Cell Line Corefrom 2015 to 2019. CRF-SB, EFE184, ETN1, KLE, HOC7, UNP251,OVCAR3, OVCAR8, and MCF7 cells were maintained in RPMI1640medium supplemented with 5% FBS and incubated at 37�C in ahumidified incubator in an atmosphere of 95% air/5% CO2. Cell lineidentities were reauthenticated by short tandem repeat (STR) DNAfingerprinting using the AmpF STR identifier kit according to themanufacturer's instructions (Applied Biosystems, catalog no.4322288). The STR profiles were compared with known ATCCfingerprints; with the Cell Line Integrated Molecular Authenticationdatabase, version 0.1.200808 (23); and with the MD Anderson fin-gerprint database. The STR profiles matched knownDNA fingerprintsor were unique. The ID8 mouse ovarian surface epithelial cells,obtained from Vahid Afshar-Kharghan (MD Anderson, Houston,TX), were maintained in DMEM (high-glucose, Cellgro) supple-mented with 4% FBS, 100 U/mL penicillin, 100 mg/mL streptomy-cin, 5 mg/mL insulin, 5 mg/mL transferrin, and 5 ng/mL sodiumselenite. All lines were tested for Mycoplasma every 6 months.MDA-MB-231 demonstrates reliable cell surface expression of PD-L1for flow cytometery (24).

AntibodiesPD-L1 (E1L3N-#13684), CIITA (#3793), IDH1 (#8137), mTOR

(7C10-#2983), Ub (P4D1-#3936), p62, AKT (40D4-2920), LDHB, LC3(D11-#3868), ATG5 (2630), GM130 (D6B1-#12480), HSP60, VPS34(D9A5-4263), b-actin (8H10D10-#3700), ATG16L1 (D6D5-8089),BECN1 (#3738), IFIH1 (#5321), IRF1 (D5E4-#8478), IRF2 (#4943),

IRF3 (#11904), STAT1 (D1K9Y-#14994), YAP1 (#15117), TRIM28(#4123), and protein A-HRP (#12991) were obtained from CellSignaling Technology; p62/SQSTM1 (610832) from BD Biosciences,HLA-Ds (sc-53302), and ERK2 from Santa Cruz Biotechnology;COG3 (11130-1-AP) from ProteinTech group; and COG7(EPR9942-ab168362) from Abcam.

Western blottingCell lysis was performed by lysing 5 � 106 cells with NP-40 lysis

buffer containing 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 0.5%NP-40, protease inhibitors (1873580001, Millipore Sigma), and phos-phatase inhibitors (4906837001, Millipore Sigma) on ice for 15minutes followed by microcentrifugation at 14,000� g for 15 minutesat 4�C to remove debris. Immunoblotting was performed using 50 mgof protein lysates resolved in SDS-PAGE, transferred to polyvinylidenedifluoride membranes (88520, Thermo Fisher Scientific), probed withprimary and secondary antibodies (A9917 and AP307P, Sigma),followed by signal detection using the ECL reagents (GERPN2019)from Sigma as instructed. Equal protein loading was verified byblotting ERK2 (tumor lines), LDHB, b-actin, mTOR, or pan AKT.

ImmunoprecipitationImmnoprecipitation was performed per the manufacturer's proto-

col (Cell Signaling Technology). Briefly, 1 mg of protein lysate wasprecleared with protein A magnetic beads (#73778) and was used forthe COG3 and IRF1 immunoprecipitation at 1:50 antibody to lysatedilution (v/v). Following overnight incubation at 4�C, the immuno-complexes were incubated with magnetic protein A beads for 20minutes at room temperature and then washed five times followedby Western blotting.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP)-PCR assaywas performed

as described previously (25). Briefly, cells were crosslinked with 1%formaldehyde for 10 minutes and the reaction was stopped by adding125 mmol/L glycine. Cells were then lysed and chromatin DNA wasfragmented using a Bioruptor Sonicator (Diagenode). The sampleswere immunoprecipitated with 4 mg of the IRF1 (#8478, Cell SignalingTechnology) antibody overnight at 4�C and a concentration-matchedrabbit IgG isotype control (#3900, Cell SignalingTechnology)was usedfor mock precipitation. The protein–DNA cross-links were thenreversed and the DNA purified and analyzed by quantitative real-time PCR on the ABI ViiA 7 Real-Time PCR system using the PowerSYBR Green PCR Master Mix (4368577, Thermo Fisher Scientific).The primers used for qPCR are as follows. Pair 1: -666 forward-TCCTTAGGGTGGCAGAATATCAG, -605 reverse-CCCATCCC-GAGCTAC-ATCTTT; Pair 2: -103 forward-TGAAAGCTTCCGCC-GATTT, -53 reverse-TGCCGGG-CGTTGGA; Pair 3: þ120 forward-TGCCCACGGCCCAGTAT, þ179 reverse-GTAGAGACCCTCCG-TCCTAAAGTG; Pair4: þ146 forward-GCTCGCTGGGCACTT-TAGG; þ205 reverse-TCCTCTCTCCATCCCAAAGAAA.

IHCTissues were first fixed in in 10% neutral buffered formalin for

24 hours, preserved in 70% ethanol, processed, and embedded inparaffin. Tissue blocks were then cut into 5-mm sections on positivelycharged slides, deparaffinized, and rehydrated. Mouse specific PD-L1(#64988, 1:200) and CD8a (#98941, 1:200) antibodies from CellSignaling Technology, and CD3e (sc-20047, 1:100) from Santa CruzBiotechnology were used for IHC using SignalStain reagents (#8112,#14746, and #8114 or #8125) per Cell Signaling Technology protocol.

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Quantitative data were obtained with ImageJ analysis of positive cellsper random area.

Flow cytometryCell surface PD-L1was stainedwith PE-conjugated PD-L1 antibody

(clone MIH1; catalog no. 12-5983-42) from Thermo Fisher Scientificfor 30 minutes at 4�C. Control staining was performed using man-ufacturer recommended mouse IgG1 isotype and treatment-matchedsamples. Data were acquired using the FACSCelesta Cell Analyzer (BDBiosciences) and analyzed using the Flowjo software (Flowjo, LLC).

RNAiON-TARGETplus siRNAs targeting human COG2 (L-019487-01-

0005), COG3 (L-013499-02-0010), ATG5 (LQ-004374-00-0005),YAP/TAZ (LQ-012200-00-0005/L-009608-00-0010), IRF3 (L-009608-00-0010), IRF1 (LQ-011704-00-0005), and IRF2 (L-011705-02-0010) were fromDharmacon. RNAi silencing was performed usingLipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Sci-entific) according to the manufacturer's protocol. Briefly, 20 nmol/LsiRNA was transfected and control cells were transfected with20 nmol/L nontargeting siRNA (Dharmacon).

Quantitative real-time PCR assayTotal RNA was isolated using the RNeasy Plus Mini Kit (Qiagen,

#74136) according the manufacturer's instructions. Briely, 5 � 106

cells were lysed and homogenized. RNA concentration was deter-mined using a NanoDrop2000c Spectrophotometer (Thermo FisherScientific) and 2 mg RNA was used for first strand cDNA synthesisusing the High Capacity cDNA Reverse Transcription kit (Invitrogen,#4368814). Real-time PCR was performed in a total volume of 20 mLper reaction in duplicates in twin.tec real-time PCR Plates (Eppendorf,#0030132718), covered with Masterclear Real-Time PCR Film(Eppendorf, #0030132947). PCR probes CD274 (Hs01125301_m1)and ACTB (Hs01060665_g1) from TaqMan Gene Expression Assays(Invitrogen) were used as instructed. Each reaction contained 40 ng ofcDNA, 10 mL of TaqMan Fast Universal PCR Master Mix (2�), noAmpErase UNG (Invitrogen, #4366073). TaqMan reactions were runon Mastercycler ep realplex (Eppendorf) with the following thermalcycling protocol: 95�C for 2 minutes followed by 40 cycles of 95�C for15 seconds, 55�C for 15 seconds, and 68�C for 20 seconds. CD274 geneexpression was quantified using the comparative Ct (2

�DDCt) methodwith Ct values normalized to the housekeeping gene (ACTB).

Cell fractionationSubcellular fractionation was performed using the FractionPREP

cell fractionation kit (K270) from Biovision according to the manu-facturer's instructions.

Cell viability assayCell viability was assayed using the CellTiter-BlueCell Viability Assay

kit (G8081) from Promega according to the manufacturer's protocol.

Reverse-phase protein microarray analysis and high-throughput drug screen

Reverse-phase protein microarray analysis (RPPA) assayswere performed in the MD Anderson CCSG core as described athttp://www.mdanderson.org/education-and-research/resources-for-professionals/scientific-resources/core-facilities-and-services/functional-proteomics-rppa-core/index.html. Briefly, serially diluted lysates werespotted onto FAST slides (Schleicher & Schuell BioSciences) using arobotic GeneTAC arrayer (Genomic Solutions, Inc.). After printing, the

slides were blotted sequentially with Re-Blot (Chemicon), I-Block,and a biotin blocking system (Dako), probed with primary anti-bodies, and incubated with biotin-conjugated secondary antibodies.The signals were then amplified using a Catalyzed Signal Ampli-fication kit (DakoCytomation) according to the manufacturer'sinstructions. The processed slides were scanned and quantitatedusing the Microvigene software (VigeneTech Inc.) and the quan-titative values of five consistently expressed proteins (p38, JNK,ERK, mTor, GSK) as internal controls. To screen for the effects onPD-L1 protein levels, a total of 40 drugs or drug combinationstargeting cancer pathways that are entering or likely to enter clinicaltrials were surveyed across established cancer cell lines and RPPAdatasets (Supplementary Table S1).

Mouse studies and in vivo tumor modelsAll animal work and protocols were supervised and approved by the

Institutional Animal Care and Use Committee of MD Anderson(Houston, TX). ID8 cells (1 � 106 in 100 mL PBS) were intraperito-neally injected into C57BL/6 mice (female, 6–8 weeks old, CRL/NCI).After transplantation, cells were allowed to grow for 1 week, and thenmice with established tumors were randomly sorted into differenttreatment groups with 5 mice/group. ID8-bearing mice were thentreated with isotype control IgG or PD-L1 antibody (200 mg/mouse in100mLPBS, B7-H1, clone 10F.9G2, BioXCell; i.p. injection) every threedays, verteporfin 60mg/kg daily in 8%DMSO (100 mL; i.p.), BMN 6730.33 mg/kg daily in 2.64% ethanol (100 mL; oral gavage), or thecombination of BMN 673 with either PD-L1 antibody or verteporfin.Tumor progression was monitored once a week using a Xenogen IVISSpectrum in vivo bioluminescence imaging system (PerkinElmer).Tumor volume was determined on the basis of the total flux (photonsper second). Mice reaching a humane endpoint or weighingmore than35 g as a result of tumor growth and/or ascites were euthanized.

Lewis lung carcinoma (LLC) cells (5 � 105 in 100 mL PBS) wereinoculated subcutaneously into C57BL/6 mice or nude (NU/J, TheJackson Laboratory; 8–12 weeks) in both flanks. Five days afterinoculation, mice were randomly divided into 4 groups (n¼ 6/group)and treated with vehicle, BMN 673 0.33 mg/kg daily (oral gavage),verteporfin 30 mg/kg daily (i.p. injection), or the combination ofBMN 673 with verteporfin for 16 days. Tumors size were measuredevery 2 days by digital calipers to determine tumor volume using theformula [length/2] � [width2].

Statistical analysisAll statistical analyses were done in GraphPad Prism 8 software.

Correlations between TRIM28 and CIITA-MHC II gene expressionwas analyzed using the linear regression test. On the basis of pilotstudies of the anti–PD-1 experiment, 5mice per groupwas sufficient toidentify the expected effectswith 90%power.Overall survival of varioustreatment groupswas analyzedusing theCox regressionmodel, and thelog-rank test was used to determine the P values. Otherwise, unpaired ttests were used to generate two-tailed P values and P < 0.05 wasconsidered statistically siginificant. TheCancerGenomeAtlas (TCGA)data were analysed using cBioPortal (cbioportal.org) and PanCancerAtlas datasets for each cancer types were used.

ResultsIdentification of verteporfin as a potent inhibitor of PD-L1expression

Using downregulation of PD-L1 as a read-out by RPPAs to assessdrugs in use or likely to enter trials with well-characterized molecular

Verteporfin Inhibits PD-L1

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targets, we performed a candidate drug screen (n ¼ 40) for inhibitionof PD-L1 expression discarding low-potency, narrow-spectrum, andupregulating candidates, which identified a single drug candidate,verteporfin. Verteporfin suppressed PD-L1 expression effectively in allsix cell lines (T-cell leukemia; B-cell leukemia; ovarian; endometriumn¼ 3; Fig. 1A; Supplementary Table S1; Supplementary Fig. S1). In anadditional panel of eight human cancer cell lines (ovarian, n ¼ 5;osteoblastoma, n¼ 1; and lung cancers, n¼ 2) and twomurine cancercell lines (ovarian and lung), verteporfin abolished basal PD-L1 proteinexpression, including differential glycosylated states as reflected by thedouble bands on Western blots (7), regardless of genetic background,lineage specificity, and basal (intrinsic) PD-L1 expression (Fig. 1A–D).Cell fractionation revealed that verteporfin decreased membrane-associated PD-L1 (functionally relevant PD-L1) in EFE184 cells(endometrial cancer; Fig. 1E) and flow cytometry showed that verte-porfin reduced PD-L1 expression on both the surface of cancer cells(Fig. 1F) and on antigen-presenting cells (Supplementary Fig. S1D).Verteporfin suppressed both IFN-induced PD-L1 protein expression(Supplementary Fig. S1B–S1D) and mRNA expression (Fig. 1G).However, in contrast to the marked loss of PD-L1 protein, verteporfinhad little effect on intrinsic PD-L1mRNA expression in the absence ofIFNg (Fig. 1H). Thus, verteporfin engaged at least two independentmechanisms to downregulate PD-L1 expression.

Verteporfin activated Golgi-related PD-L1 autophagyAs verteporfin inhibits autophagy (26), we evaluated whether

autophagy was required for verteporfin-induced loss of PD-L1.Although verteporfin exhibited a modest dose-dependent growth-inhibitory effect on EFE184 cells, cotreatment with chloroquine, whichinhibits the final step of autophagy, led to an approximately 16-foldincrease in verteporfin-induced growth suppression and amarked lossof cell viability (Fig. 2A); these data were consistent with a crucial roleof autophagy in maintenance of viability of cells treated with verte-porfin. Transmission electron microscopy (TEM) revealed a markedincrease in autophagosomes in the cells treated with verteporfin, andaltered Golgi apparatus with swollen and disrupted structures(Fig. 2B). The morphologically disrupted Golgi networks were inproximity to autophagosomes. Consistent with the TEM data, verte-porfin treatment led to progressive loss of high molecular weightubiquitinated proteins in a dose-dependent manner, increased LC3lipidation (LC3 1 and II), and loss of the selective autophagy substrateand adaptor p62/SQSTM1 (Fig. 2C and D), indicating active autop-hagy consistent with a prior report (27).

In addition to loss of p62 and PD-L1, verteporfin induced a markeddecrease in the Golgi proteins COG3, COG7, and the Golgi matrixprotein GM130 (Fig. 2D). Chloroquine treatment abrogated verte-porfin-induced loss of PD-L1, p62/SQSTM1, and Golgi proteins(Fig. 2D). These data suggested that verteporfin induced autop-hagy-mediated degradation of the Golgi apparatus, which was likelya consequence of verteporfin-induced organelle damage. Indeed, wefound COG3 physically associated with VPS34 (a crucial membranecomponent of the autophagy machinery), the autophagy adaptormolecule p62, and GM130 (Fig. 2E). Next, we interrogated whetherthe lobe A subunits might have a role in autophagy-dependent PD-L1removal in cancer cells. RNAi-mediated gene silencing was performedfor COG2 and COG3. The COG2 RNAi led to cross depletion ofCOG3, consistent with the finding that both COG3 and COG2 arerequired for stabilizing lobe A complexes (28). Nonetheless, RNAi ofeither COG2 or COG3 increased intrinsic PD-L1 expression, withCOG2 depletion having a stronger effect (Fig. 2F), consistent with arole for the oligomeric Golgi complex in regulating PD-L1 expression

as a quality control mechanism. However, depletion of COG3 but notCOG2 attenuated verteporfin-induced loss of PD-L1 (Fig. 2F), sug-gesting a crucial role for COG3, but not COG2, in verteporfin-inducedPD-L1 removal.

Verteporfin inhibited IRF1-dependent PD-L1 transcriptionSupporting a role for autophagy in mediating verteporfin-induced

loss of PD-L1, RNAi-mediated knockdown of ATG5, a gene that isessential for canonical autophagy (29), mitigated the effect of verte-porfin on PD-L1 downregulation (Fig. 3A). Despite a role for autop-hagy in downregulation of PD-L1 protein expression, verteporfin wassufficient to decrease PD-L1 expression in ATG5-depleted cells atincreased concentrations (1 mmol/L vs. 0.5 mmol/L). At higher drugconcentrations, verteporfin abolished IFN-induced PD-L1 expressionin the presence of chloroquine (Fig. 3B), suggesting that chloroquine-sensitive autophagy was dispensable for loss of PD-L1 in this setting.Because verteporfin treatment also resulted in significant downregula-tion of PD-L1 mRNA expression (Fig. 1G), we explored mechanismscontrolling PD-L1 gene expression.

Verteporfin inhibits YAP/TAZ function and YAP1 inhibits IRF3signaling (30). Thus, RNAi was performed to interrogate the roles ofthe YAP1, TAZ (WWTR1), IFIH1, and IRF3 cascade in associationwith PD-L1 expression. Knockdownof these genes hadminimal effectson both the basal expression of PD-L1 or IFN-induced PD-L1 expres-sion, with depletion of YAP1 and TAZ (WWTR1) leading to increasedrather than decreased PD-L1 in the presence of IFNg (SupplementaryFig. S2). Thus, the effect of verteporfin on PD-L1 expression is unlikelyto be attributed to the YAP/TAZ signaling cascade.

Given the ability of verteporfin to abolish IFN-induced PD-L1expression, we next sought to determine whether verteporfin actedto disrupt PD-L1 gene transcription. Both the signal transducer andactivator of transcription 1 (STAT1) and the main transcription factorIRF1, downstream of the IFNg receptor, are essential for IFN-inducedPD-L1 gene expression (31). Indeed, IFN increased IRF1 expression ina panel of cancer cell lines, and IRF1 protein expression directlycorrelated with PD-L1 levels (Supplementary Fig. S3). IRF1 depletionabolished IFN-induced expression of PD-L1 (Fig. 3C), whereas IRF2knockdown had no effect despite a possible role of IRF2 in eitheropposing or complimenting that of IRF1 (32). Thus, we examined theeffect of verteporfin on PD-L1mRNA in the context of IRF1 depletion.Verteporfin markedly decreased PD-L1 mRNA expression, and IRF1depletion had a stronger effect, whereas verteporfin did not causedecreases in PD-L1 mRNA expression in the setting of IRF1depletion (Fig. 3D), suggesting that verteporfin acted via inhibitionof IRF1-dependent PD-L1 transcription. However, IFN-inducedSTAT1 and IRF1, including their nuclear localization, were notaffected by verteporfin (Fig. 3E), suggesting intact IFN-STAT1signaling upstream of IRF1.

To determine where in the STAT1 pathway verteporfin might beacting, we performed IRF1 immunoprecipitation and found thatwhereas IFN-g induced amarked increase in IRF1–STAT1 association,verteporfin treatment led to complete disruption of the IRF1–STAT1complex (Fig. 3F). We then performed ChIP assays to determine theeffect of verteporfin on IFN-induced IRF1-binding to PD-L1 genepromoter regions (Fig. 3G). Indeed, IRF1-binding could not bedetected in unstimulated cells, but IFNg induced marked increasesin the binding of IRF1 to the PD-L1 promoter (Fig. 3H). Strikingly,despite inhibiting PD-L1 expression, verteporfin treatment led to amodest increase in IRF1 binding in the absence of IFN, and a markedincrease was detected in the presence of IFN (Fig. 3F). The paradoxicalincrease in IRF1 binding to the PD-L1 promoter after verteporfin

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E

Figure 1.

Verteporfin decreased intrinsic and IFN-induced PD-L1 expression. A, Summarized data of PD-L1 protein expression (fold change relative to untreated baseline) asdetermined by Western blot analysis in human ovarian (ES2, HOC1, and UPN251), osteoblastoma (U2OS), and lung (H226 and H358) cancer cells treated with(þ) or without (�) verteporfin (1 mmol/L, 24 hours), as shown in B and C. Western blots for PD-L1 expression in human ovarian cancer cells (B) and lung cancercells (C). D, Murine cancer cells treated with (þ) or without (�) verteporfin and IFNg . E, Western blots of PD-L1, a loading control (mTOR), membranefractionation (CD74), and cytosolic fraction (IDH1) of EFE184 cells (endometrial carcinoma) treated with (þ) or without (�) IFNg (10 ng/mL) and verteporfin(1 mmol/L, 24 hours). Data represent two or more independent experiments. F, Flow cytometry analysis of PD-L1 expression on the surface of MDA-MB-231(breast carcinoma) cells treated with (þ) or without (�) verteporfin (2 mmol/L) for 24 hours. Relative PD-L1 mRNA expression in EFE184 cells treatedwith or without verteporfin for 24 hours either in the presence (G) or absence (H) of IFNg (5 ng/mL). Data represent mean � SD of three independentexperiments. ��, P < 0.001 (two-tailed t test). VP, verteporfin.

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treatment appeared to suggest futile recruitment of IRF1, probably as aresult of disruption of the IRF1–STAT1 protein complex. Indeed,additional ChIP assays showed that verteporfin decreased the recruit-ment of STAT1 to the PD-L1 promoter in either the absence orpresence of IFN (Supplementary Fig. S4). Taken together, our datasuggested that verteporfin disrupts IFN-induced IRF1–STAT1 inter-action, leading to suppression of PD-L1 transcription with nonpro-ductive trapping of IRF1 to the PD-L1 promoter.

Differential effects of verteporfin on IRF1-dependenttranscription

In addition to loss of PD-L1 expression, IRF1 knockdown also ledto a marked decrease in the CIITA transcription factor and itsdownstream CD74 protein (Fig. 4A). The MHC II cascade branchof IRF1 signaling was not inhibited in verteporfin-treated cells in thepresence of IFN (Fig. 4B), suggesting that verteporfin blocked theIFN–IRF1–PD-L1 axis specifically but had little effect on IRF1-CIITA.Such pathway specification might allow the PD-L1 immune check-point pathway to be blocked without collateral suppression of CIITA-

dependent tumor immunogenicity. We next sought to understand theunderlying mechanism behind this. TRIM28 is a cofactor acting eitherto activate or repress target gene transcription (33). Although previ-ously reported in association with IRF1 (34), the role of TRIM28 inregulating IRF1 signaling remains unclear. We found in parallel toSTAT1, that the TRIM28–IRF1 interaction was markedly increased inresponse to IFN and that the interaction was abrogated by verteporfin(Fig. 3F). Strikingly, shRNA-mediated TRIM28 depletion led tomarked increases in CIITA andHLA-D (Fig. 4C), suggesting TRIM28normally acted to inhibit the CIITA-MHC II cascade in a feed-forwardmanner that is responsive to IFN.

Indeed, analyses of TCGA data revealed that TRIM28 expressioninversely correlated with CIITA-MHC II gene expression andCD74 inthe majority of human cancer types that span immunologic reactivity(Supplementary Fig. S5A and S5B). Gene expression profiling revealedtwo distinct tumor clusters in lung cancer, conforming to high and lowCIITA signature classes of gene expression, respectively (Supplemen-tary Fig. S5C), which in turn exhibited a very strong (P¼ 9.27� 10�16)inverse association with TRIM28 mRNA expression (Supplementary

GM130

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Figure 2.

Autophagy was required for cell survival,and verteporfin-induced loss of PD-L1. A, Celldensity after treatment with either verteporfin(0–1,000 nmol/L) or chloroquine (CQ, 0–20 mmol/L) alone or the combination of the twodrugs for 6 days. B, Autophagosomes asrevealed by TEM in EFE184 cells treated with(þ) or without (�) verteporfin (1 mmol/L) for24 hours. Red arrows, Golgi; yellow arrows,autophagosomes. Data represent mean � SDfrom 30 cells. �� , P < 0.001 (two-tailed t test).C, Western blots of ubiquitin (Ub) and p62/SQSTM1 (p62) in EFE184 cells treated with ver-teporfin (0–3 mmol/L). AKT and LDHB wereblotted for loading control. D, Western blotanalyses of protein expression in EFE184 cellstreated with (þ) or without (�) verteporfin(0.5 mmol/L) and chloroquine (10 mmol/L) for96 hours. Quantification data (densitometryvalues normalized to lane 1) are provided belowrespective blots. E, Western blots for proteinspresent in COG3 immunoprecipitates fromEFE184 cells treated with (þ) or without (�)verteporfin (1 mmol/L) and chloroquine(10 mmol/L) for 24 hours. F, Western blots forprotein expression in EFE184 cells treated with(þ) or without (�) verteporfin for 24 hours aftertransfection of nontargeting (siNT), COG2(siCOG2), or COG3 (siCOG3) siRNA for 48hours.Densitometry data (fold change) of PD-L1expression are presented below the blot.HSP60, AKT, and LDHBwere blotted for loadingcontrol. Western blotting data represent twoor more independent experiments. CQ, chloro-quine; VP, verteporfin.

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A

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Figure 3.

IRF1 was essential for IFN-induced PD-L1, and verteporfin disrupted IRF1 complex.A,Western blots for protein expression in EFE184 cells treated with (þ) or without(�) verteporfin for 24 hours after transfection of nontargeting (siNT) or ATG5 (siATG5) siRNA for 48 hours.B,Western analysis of protein expression in EFE184 cellstreated with (þ) or without (�) verteporfin (2 mmol/L), chloroquine (CQ; 20 mmol/L), and IFNg (5 ng/mL) for 24 hours. C,Western blots for protein expression inEFE184 cells treatedwith (þ) orwithout (�) IFNg (5 ng/mL) for 24 hours after transfection of nontargeting (siNT), IRF1 (siIRF1), or IRF2 (siIRF2) siRNA for 48 hours.D,qPCR for PD-L1 mRNA expression in EFE184 cells treated with (þ) or without (�) verteporfin (mmol/L) and IFNg for 24 hours after transfection with nontargeting(siNT) or IRF1 (siIRF1) siRNA for 48 hours. Data represent mean � SD (n ¼ 6). � , P < 0.05; �� , P < 0.001; N.S., not significant (two-tailed t test). E,Western blots forproteins after subcellular fractionation in EFE184 cells treated with (þ) or without (�) verteporfin (1 mmol/L) and IFNg (5 ng/mL) for 24 hours.Western blotting datarepresent two or more independent experiments. F, Western blots for proteins present in IRF1 immunoprecipitates (IP: IRF1) and mock immunoprecipitation withnormal rabbit IgG (NR-IgG) from EFE184 cells treated with (þ) or without (�) IFNg and verteporfin for 24 hours. Blots for input and IRF1-immunodepleted (IRF1-de)lysates are shown on the right. PD-L1 gene (CD274) promoter regions (R1�4; G) and CHIP assays for IRF1 (H) and STAT1 (Supplementary Fig. S4) recruitment inEFE184 cells treated with or without IFNg and verteporfin for 24 hours. VP, verteporfin.

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Fig. S5D–S5F). Thus, the role of TRIM28 in suppressing the IRF1–CIITA pathway appeared to be generalizable.

Importantly, higher TRIM28 expression was associated with poorerpatient outcomes inmelanoma and lung cancer (Fig. 4D andE), which

are considered immunologically reactive, with a demonstrated benefitto immune checkpoint inhibition (35). To determine whether thestatistical association between TRIM28 and patient outcomes mightsimply reflect a noncausal correlation, we analyzed the effect of a

IFNVP

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Figure 4.

TRIM28 suppressed the CIITA-MHC II cascade downstream of interferon signaling. A, Western blots for interferon target proteins in EFE184 cells treated with (þ) orwithout (�) IFNg (5 ng/mL) for 24 hours after transfection of nontargeting (siNT), IRF1 (siIRF1), or IRF2 (siIRF2) siRNA for 48 hours. B, Western blots for proteins inEFE184 cells treatedwith (þ) orwithout (�) IFNg (5 ng/mL) and verteporfin (VP;0.5mmol/L) for 24hours. Quantificationdata (densitometry values normalized to lane 1)are provided below respective blots. Data represent two or more independent experiments. C, Western blot analyses of protein expression in control (sh-CTL) andTRIM28 knockdown (sh-TRIM28) MCF7 cells treated with (þ) or without (�) verteporfin (1 mmol/L) and IFNg (5 ng/mL) for 24 hours. Quantification data(densitometry values normalized to lane 1) are provided below respective blots. Kaplan–Meier estimates of overall patient survival of melanoma (P ¼ 0.0065,n ¼ 287; D) and lung cancer (P ¼ 0.0022, n ¼ 230; E) expressing high and low expression of TRIM28 mRNA and melanoma (P ¼ 0.0049, n ¼ 287; F), lungadenocarcinoma (P ¼ 0.0015, n ¼ 230; G), breast cancer (P ¼ 0.0104, n¼ 963; H), ovarian cancer (P ¼ 0.048, n ¼ 182; I), stomach cancer (P ¼ 0.0167, n¼ 369;J), and glioblastoma multiforme (P ¼ 0.565, n ¼ 136; GBM; K) expressing high and low expression of CIITA mRNA from the TCGA database. P values < 0.05were considered statistically significant as determined by log-rank tests. GBM, glioblastoma multiforme; LUAD, lung adenocarcinoma.

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downstream event, that is, the expression of CIITA.We found that themRNA levels of CIITA were indeed positively associated with morefavorable patient outcomes, with high statistical power across a broadspectrum of immunologically reactive human cancers albeit notnecessarily in immunologically nonreactive tumors such as GBM(Fig. 4F–K), suggesting that the effects of TRIM28 and CIITA onpatient outcomes might be mechanistically linked in specific cancerlineages.

Verteporfin mediated PD-L1 blockade in syngeneic tumormicroenvironments

We next hypothesized that therapeutic interventions that acti-vate the CIITA-MHC immunogenicity cascade, which usually alsodrive PD-L1 expression, would synergize with verteporfin. BecausePARP inhibitors synergize with immune checkpoint blockade(36), we sought to determine whether PARP inhibition might affectthe CIITA-MHC cascade. We found that PARP1 copy numberincreases in over 85% of lung cancers (adenocarcinomas), whichcorrelated with PARP1 mRNA expression (Fig. 5A; Pearson r ¼0.511). Higher PARP1/2 mRNA expression was associated with

significantly poorer overall and disease-free survival in patientswith lung cancer and with decreased mRNA expression ofCIITA-MHC and PD-L1 genes (Fig. 5B–G).

PARP inhibitors have activity across a spectrum of cancers andare approved for first-line treatment of BRCA-mutant ovariancancers (37). Notably, murine ID8 ovarian carcinoma cells exhib-ited little sensitivity to verteporfin in cell culture and no synergismwith the BMN 673 PARP inhibitor (Fig. 6A). Given these prop-erties, we tested the in vivo therapeutic effects of verteporfinmonotherapy or in combination with BMN 673 in establishedimmune competent mice bearing ID8 cells intraperitoneally indirect comparison with PD-L1 antibody (Fig. 6B). Monotherapywith either verteporfin or PD-L1 had a modest effect on the survivalof ID8-burdened mice (Fig. 6B), consistent with previous studiesshowing these cells to have modest intrinsic immunogenici-ty (38, 39). In contrast, the combination of verteporfin and BMN673 improved outcome compared with either monotherapy that wasequivalent to the combination of PD-L1 and BMN 673 (Fig. 6B).Independent of BRCAmutations, PARP inhibition induces immuneresponses in cancer models including that of lung cancer, which has

mR

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Figure 5.

PARP1 expressionwas associated with PD-L1 expression and the CIITA cascade. Correlation of PARP1 mRNA expression with putative PARP1 gene copy number (A),disease-free survival (B), and overall survival (C) of patients with lung adenocarcinoma (n ¼ 230, TCGA database). P < 0.05 as determined by log-rank tests.Enrichment analysis for the association of CIITA (D), MHC (E and F), and PD-L1 (G) expression with high and low expression of PARP1/2 mRNA expression in lungadenocarcinoma (TCGA database). P values < 0.05 were considered statistically significant as determined by two-tailed unpaired t tests.

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been exploited for therapeutic intervention (40, 41). Similar to ID8cells, murine LLC cells exhibited limited sensitivity to verteporfinand no synergism with the BMN 673 PARP inhibitor in vitro(Fig. 6C). Monotherapy with either verteporfin or BMN 673 hada modest effect on tumor growth in a syngeneic setting, whereas thecombination of verteporfin and BMN 673 produced a significantreduction in tumor volume (Fig. 6D). In LLC tumors, verteporfintreatment led to marked decreases in PD-L1 expression andincreases in CD8þ T cells, especially in the vereporfin and BMN 673treatment group (Fig. 6E; Supplementary Fig. S6A and S6B). Incontrast, verteporfin and BMN 673, either administrated alone or in

combination, were not effective in immunodeficient nude micerelative to control (P > 0.1 one-way ANOVA; Fig. 6F), emphasizingthe role of immune responses in the drug action. The difference intumor size on day 21 of the verteporfin-treated mice was notstatistically significant.

PARP inhibition induced PD-L1 expression on high endothelialvenules

While T-cell infiltration was consistent with the overall outcome ofPD-L1 suppression, we observed that PD-L1 expression was highlyheterogeneous in the LLC tumor microenvironment. The strongest

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Figure 6.

Verteporfin and PARPi inhibited syngeneic ovarian cancer and lung carcinoma cell growth. A, Dose–response curves for relative viability of mouse ovarian cancercells (ID8) treated with or without VP, BMN 673, or the combination of both for 72 hours in standard cell culture. A–F ¼ 0, 0.012, 0.04, 0.12, 0.37, and 1.1 mmol/L,respectively. Data represent mean� SD (n¼ 9). B, Kaplan–Meier survival estimates of C57/BL6 mice intraperitoneally implanted with ID8 cells (N¼ 5). Establishedtumors were treated with vehicle (control), PD-L1, BMN 673, verteporfin, or the combination of BMN 673 with either PD-L1 or verteporfin. � , P < 0.05 (log-rank test).C, Dose–response curves of verteporfin in combination with BMN 673 for relative viability of LLC cells. D, Growth curves of LLC in C57BL/6 mice treated with orwithout verteporfin (30mg/kg), BMN673, or the combination of the twodrugs. � ,P<0.05; �� ,P<0.01 (two-tailed t test).N¼8 tumors per group. The experimentwasrepeatedwith similar results.E, IHC staining (summarized data) of PD-L1 andCD8A in LLC tumors treatedwith orwithout verteporfin, BMN673, or the combination ofthe twodrugs. Data represent average�SDof 13 areas of 300nuclear events. � ,P<0.05; �� ,P<0.01 (two-tailed t test). The controlwas used as the baseline and set at1. Supplementary Figure S6 shows representative IHC images. F,Growth curves of LLC in nudemice treatedwith or without verteporfin (30mg/kg), BMN 673, or thecombination of the two drugs. VP, verteporfin.

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PD-L1 signal was detected in the tumor vasculature in BMN 673–treated tumors (Supplementary Fig. S6A). Although PARP inhibitiondid not affect tumor cell expression of PD-L1 (SupplementaryTable S1). We then sought to determine whether tumor-associatedendothelial cells were the sources of PD-L1 expression in the BMN673-treated tumors. Although PD-L1 expression appeared to beassociated with LYVE1-positive lymphatic vasculature (Supplemen-tary Fig. S6B), closer examination only revealed a relatively weakexpression of PD-L1 in LYVE1-positive cells.

Because high endothelial venules (HEV), which are specializedpost-capillary structures, are the only known routes of tumorinfiltration of lymphocytes (42, 43), we tested the hypothesis thatPARP inhibition drives PD-L1 expression in HEVs. PARP inhibi-tion with BMN 673 on lymphatic endothelial cells led to increasedprotein expression of CIITA and PD-L1 (Fig. 7A). Whereas PARPi-induced PD-L1 expression was suppressed by verteporfin, CIITAexpression was not significantly altered even at supraphysiological

drug doses (i.e., ≥1 mmol/L; Fig. 7B). Ex vivo analysis of tumorsstained for the selective HEV marker MECA-79 formed poorlydeveloped vascular-like structures in control tumors but highlyorganized structures were detected in BMN-673–treated tumorswith strong luminal MECA-79 staining (Fig. 7C). Strikingly, despitea weak coexpression of PD-L1 and MECA-79 in control tumors,strong PD-L1 expression was detected in MECA-79–positive cells inBMN 673–treated tumors (Fig. 7D), suggesting that PARP inhibi-tion induced PD-L1 expression in HEVs. PD-L1 expression on theHEVs thus inhibited effector and proliferating T cells as they exitedfrom the peripheral circulation into the tumor microenvironment(Fig. 7E).

DiscussionHigh-throughput screening identified verteporfin as a lead candi-

date for inhibiting PD-L1, suggesting the potential repurposing of the

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Figure 7.

PD-L1 expression on high endothelial venulesand the effects of PARPi and verteporfin. Aand B, Western blots for CIITA and PD-L1protein expression in SVEC4.10 (murine lym-phatic endothelial) cells treated with BMN673 (0–100 nmol/L for 96 hours; A) or BMN673 (100 nmol/L for 96 hours) and vertepor-fin (0–2 mmol/L for 48 hours; B). Data rep-resent two or more independent experi-ments. C and D, Immunofluorescent stainingfor MECA-79, a selective marker for HEVs,and PD-L1 in syngeneic LLC tumor treatedwithout (�) or with (þ)BMN 673. Scale bars,20 mm. E, Schematic of the mechanismunderlying the synergy between PARP inhi-bition and verteporfin-mediated PD-L1 block-ade. PARP inhibition promoted HEV neo-genesis and PD-L1 expression, including onHEVs, whereas verteporfin abrogated PD-L1expression through Golgi-related autophagyand suppression of IRF1-dependent tran-scription. The combination of PARP inhibitionand PD-L1 blockade produced a therapeuticsynergy. VP, verteporfin.

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FDA-approved ophthalmological drug verteporfin for oncologic ther-apeutic benefit. Verteporfin is a photosensitizer commonly prescribedfor retinopathy but also has antitumor properties in various experi-mental settings (44, 45). Verteporfin possesses intrinsic antitumoractivities through targeting YAP1 and other mechanisms (44, 45).Given the patient safety profile and relative low cost ($1700 perinjection, wholesale), verteporfin could be considered as an alternativeapproach to PD-L1 blockade. PD-L1 blockade has therapeutic benefitin non–small cell lung carcinoma in a randomized phase III study (46).Notably, the patients with lung cancer with the highest expression ofPD-L1 derived the greatest benefit from PD-L1. PARP inhibitors, suchas BMN 673, upregulated PD-L1 on HEVs and the combination ofPARP inhibitors and PD-L1 blockade increased the therapeutic effectsin vivo. Our preclinical data may provide a strong rationale for thecombinatorial use of verteporfin and a PARP inhibitor in patients withlung cancer, especially given the high frequency of PARP1 gain in lungcancer. Inmany other cancers such as GBM, PD-L1 is low (47) and thelack of immune effector responses in the tumor microenvironmentmay not necessarily benefit from this strategy.

Our study revealed two unique mechanisms of action of verteporfinon PD-L1 expression; an autophagy-dependent mechanism mediatedby the oligomeric Golgi complex of the COG proteins (specificallyCOG2 and COG3) for the removal of intrinsically expressed PD-L1and suppression of IFNg-induced PD-L1 expression. This selectivedownregulation of PD-L1 with verteporfin maintained the proinflam-matory IRF1–CIITA–MHC II signaling cascade. IRF1 is a key regu-latory factor for IFNg , TLR, and TNF (48–50); thus, IRF1 plays animportant role in inflammatory responses. Indeed, IRF1 knockout inmice leads to a multifaceted defect in immune surveillance (51).Depending on the specific target gene, IRF1-mediated transactivationcan occurwith orwithout its cofactor STAT1 (52). In the case of PD-L1expression, both IRF1 and STAT1 are essential (4). We showed thatverteporfin disrupted the IRF1–STAT1 interaction, thereby suppres-sing IFNg-induced PD-L1 transcription, but it increased IRF1 bindingto the PD-L1 promoter in response to IFNg . It remains unclear as towhether or not this promoter-trapping effect is required for suppres-sion of PD-L1 transcription in addition to loss of STAT1 binding.Supporting this concept, IRF1 is involved in target gene silencing (48).Our study also unveiled a previously unrecognized role for TRIM28 ininhibiting the IRF1–CIITA–MHC II axis but not PD-L1 expression.Decommissioning of this role of TRIM28 upon verteporfin treatmentprovided another layer of selectivity, allowing precision targeting of theIRF1–PD-L1 axis. More studies are needed to determine whetherTRIM28 contributes to the development of resistance to adaptiveimmune surveillance in human cancer and whether TRIM28 repre-sents a therapeutic target for reversing the immunoediting processassociated with suppression of the CIITA-MHC genes (53).

We also found that the PARP inhibitors induced PD-L1 expressionon specialized tumor-associated endothelial structures known as theHEVs, the “gateway” for tumor-infiltrating lymphocytes (43). Thepresence of HEVs correlate with more favorable patient outcomes inmultiple cancer types (54). As shown in other tumor-associatedendothelial cells (55, 56), HEV expression of PD-L1 was probablymodulating the effector and proliferative function of the T cells as theymove from circulation into the tumor microenvironment. This mech-anism would be in addition to the previously documented roleof PARP inhibition, the accumulation of double-strandedDNAbreaksin cancer cells with increases in mutation loads or the presence ofdamaged chromosomal DNA in the cytoplasm triggering the cGAS-innate immune response pathway (57). STING pathway activation inthis context also likely drives the robust T-cell infiltration we observed

in the tumors (40). We found a strong inverse correlation between themRNA expression of PARP1/2 and the proinflammatory CIITA-MHC cascade genes in multiple human cancers. Thus, PARP inhibi-tion was likely to activate the CIITA-MHC cascade, immunogenicity,and T-cell priming through multiple pathways, which consequentlyelicited immune responses and adaptive PD-L1 expression.

Despite the strong induction of both HEV neogenesis and PD-L1 inresponse to PARP inhibition, different mechanisms might be involvedin these processes. In addition to DNA repair, PARP1 regulatesgene expression through multiple mechanisms (58) and can act asa cofactor of multiple transcriptional regulators including NFkB andTNFa (59, 60). Indeed, the expression of a number of NFkB-dependent genes are impaired in PARP1�/� cells as well as in responseto PARP inhibition (61). Whereas the lymphotoxin–b-LTBR pathwayis essential for HEV neogenesis, the signaling of the closely relatedcytokine TNFa has the opposite role in experimental melanoma (62).Future studies will be directed at ascertaining the applicability ofmodulating HEV PD-L1 expression across cancer lineages and itsrelevance as a biomarker for response to PD-L1 strategies.

Enrichment markers for response to PD-L1 strategies have not hadhigh predictive value for therapeutic response, which is likely thescenario for responses to verteporfin as it may also have directantitumor activities through targeting YAP1 (44, 45). At the dosagerange that abrogated PD-L1 expression used in our study verteporfinhad little effect on either YAP1 expression or its phosphorylation, andexhibited minimal cell-autonomous cytotoxicity on cancer cells. Ver-teporfin in combination with PARP inhibition produced a strongsynergistic effect in syngeneic models of both ovarian and lung cancersbut not in cell culture, suggesting a therapeutic efficacy independent ofcell-autonomous anticancer activity. Nevertheless, verteporfin mayprove uniquely effective in cancers that rely on oncogenic signals fromYAP1. In addition, the striking coexpression pattern of MECA-79 andPD-L1 suggested that HEV staining could be considered as a potentialbiomarker for PD-L1 blockade, especially with a combination of PARPinhibitors and verteporfin within the context of a clinical trial.

Disclosure of Potential Conflicts of InterestH. Liang is a consultant/advisory board member for and has ownership interest

(including patents) in Precision Scientific. G.B. Mills is a scientific advisory boardmember for AstraZeneca, Chrysalis, ImmunoMET, Ionis, Lilly, PDX Pharma,Signalchem Lifesciences, Symphogen, and Tarveda and has ownership interest(including patents) in ImmunoMET, Signalchem Lifesciences, Tarveda, CatenaPharmaceuticals, and Spindletop Ventures. No potential conflicts of interest weredisclosed by the other authors.

DisclaimerThe funding agencies had no role in the data analysis, interpretation of the results,

or writing of this article.

Authors’ ContributionsConception and design: J. Liang, L. Wang, C. Wang, Y. Lu, J. Chen, G.B. Mills,A.B. HeimbergerDevelopment of methodology: J. Liang, L. Wang, C. Kassab, K.J. Jeong, Y. LuAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): J. Liang, L. Wang, J. Shen, B. Su, D. Fang, C. Kassab, Y. Lu, Z. Zhou,C. Lu, Z.-X. Xu, Q. Yu, S. Shao, X. Chen, M. GaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Liang, L. Wang, J. Shen, C. Kassab, K.J. Jeong,W. Zhao, Y. Lu, A.K. Jain, H. Liang, S.-C. Sun, X. Chen, F.X. Claret, Z. Ding, P. ChenWriting, review, and/or revision of the manuscript: J. Liang, L. Wang, D. Fang,C. Kassab, K.J. Jeong, S.-C. Sun, M.C. Barton, G. Peng, G.B. Mills, A.B. HeimbergerAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): L. Wang, Y. Lu, M. Gao, J. Chen, P. Chen, G.B. Mills,A.B. Heimberger

Liang et al.

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Study supervision: J. Chen, M.C. Barton, G. Peng, G.B. Mills, A.B. HeimbergerOther (dosing the animals for the study): A.L. MarisettyOther (provided RPPA analysis on this study): Y. Lu, Q. YuOther (provided the plasmidDNAmaterials and expertise in data interpretation):A.K. Jain

AcknowledgmentsThe authors thank Kenneth Dunner Jr at the High Resolution Electron

Microscopy Facility for assistance in performing electron microscopy; DavidM. Wildrick, PhD, for editorial assistance; and Audria Patrick for assisting inmanuscript preparation. This project was supported by the Gynecologic SPORE(5P50CA098258, NIH/NCI; to G.B. Mills), the Provost Retention Fund and the

Brockman Foundation (to A.B. Heimberger), and the Shanghai Pujiang Program(17PJ1401400; to C. Wang). This research was performed in the Flow Cytometry& Cellular Imaging Facility, which is supported in part by the NIH through MDAnderson's Cancer Center Support Grant CA016672.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 6, 2019; revised October 7, 2019; accepted April 2, 2020;published first April 7, 2020.

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