Translational Cancer Mechanisms and Therapy

Regorafenib Promotes Antitumor Immunity viaInhibiting PD-L1 and IDO1 Expression inMelanomaRui-Yan Wu1, Peng-Fei Kong1, Liang-Ping Xia1,2, Yun Huang1, Zhi-Ling Li1,Yun-Yun Tang1, Yu-Hong Chen1, Xuan Li1, Ravichandran Senthilkumar1,Hai-Liang Zhang1, Ting Sun3, Xue-Lian Xu1, Yan Yu1, Jia Mai1, Xiao-Dan Peng1,Dong Yang1, Li-Huan Zhou1, Gong-Kan Feng1, Rong Deng1, and Xiao-Feng Zhu1

Abstract

Purpose: Immune checkpoint blockade (ICB) therapyinduces durable tumor regressions in a minority of patientswith cancer. In this study, we aimed to identify kinase inhi-bitors that were capable of increasing the antimelanomaimmunity.

ExperimentalDesign: Flowcytometry–based screeningwasperformed to identify kinase inhibitors that can block theIFNg-induced PD-L1 expression in melanoma cells. The phar-macologic activities of regorafenib alone or in combinationwith immunotherapy in vitro and in vivowere determined. Themechanisms of regorafenib were explored and analyzed inmelanoma patients treated with or without anti–PD-1 usingThe Gene Expression Omnibus (GEO) and The CancerGenome Atlas (TCGA) datasets.

Results: Through screening of a kinase inhibitor library, wefound approximately 20 agents that caused more than half

reduction of cell surface PD-L1 level, and regorafenib was oneof the most potent agents. Furthermore, our results showedthat regorafenib, in vitro and in vivo, strongly promoted theantitumor efficacy when combined with IFNg or ICB. Bytargeting the RET–Src axis, regorafenib potently inhibitedJAK1/2–STAT1 and MAPK signaling and subsequently atten-uated the IFNg-induced PD-L1 and IDO1 expression withoutaffecting MHC-I expression much. Moreover, RET and Src co-high expression was an independent unfavorable prognosisfactor in melanoma patients with or without ICB throughinhibiting the antitumor immune response.

Conclusions: Our data unveiled a new mechanism ofalleviating IFNg-induced PD-L1 and IDO1 expression andprovided a rationale to explore a novel combination of ICBwith regorafenib clinically, especially in melanoma withRET/Src axis activation.

IntroductionImmune checkpoint blockade (ICB) therapies that unleash

dynamic and complex immune responses have providedpatients with cancer with novel treatments. Agents that inter-rupt immune checkpoints, such as anti–CTLA-4, anti–PD-1,and anti–PD-L1, have the potential to elicit durable control ofcancer and even cures; however, more than half of patientsdo not respond to ICB, and approximately 30% relapse (1).

Therefore, there are unknown mechanisms that limit the anti-tumor efficacy of ICB, and overcoming the resistance is a majorchallenge.

Commonly, when tumor antigen-specific T cells are activat-ed by ICB, IFNg is secreted and elevated in the tumor envi-ronment (2), creating an extraordinarily complex and crucialsignaling network. On one hand, IFNg exhibits antitumoreffects, including upregulation of pathogen recognition, anti-gen processing and presentation, inhibition of tumor cellproliferation, and induction of apoptosis (3, 4). On the otherhand, IFNg also shows a feedback suppression effect on anti-tumor immune response by inducing expression of a series ofimmunosuppressive factors, including PD-L1 and indolea-mine 2,3-dioxygenase (IDO; refs. 3, 5, 6). Moreover, despitethe PI3K/AKT and MAPK pathway activation (7), MYC over-expression (8), CDK5 activation (9) leading to constitutivePD-L1 expression, and ONC, PAMP, IL6 signaling pathwaysfacilitating IDO1 expression (10), the adaptive PD-L1 andIDO1 expression induced by IFNg appears to be more com-mon and potent in tumor cells as well as other tumor micro-environment immune cells (2, 3). It has been reported thatsome patients with PD-L1–positive expression may notrespond to the ICB (11). Importantly, IDO1 contributes tothe suppression of tumor immunity, and blocking the IDO1helps in controlling tumor growth (12). These observationssuggest that feedback PD-L1 and IDO1 expression inducedby IFNg may attenuate the therapeutic efficacy of ICB, and

1State Key Laboratory of Oncology in South China, Collaborative InnovationCenter for Cancer Medicine, Guangdong Key Laboratory of NasopharyngealCarcinoma Diagnosis and Therapy, Sun Yat-sen University Cancer Center,Guangzhou, China. 2Department of the VIP Region, Sun Yat-sen UniversityCancer Center, Guangzhou, China. 3Key Clinical Laboratory of Henan Province,Department of Clinical Laboratory, The First Affiliated Hospital of ZhengzhouUniversity, Zhengzhou, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

R.-Y. Wu, P.-F. Kong, L.-P. Xia, and Y. Huang contributed equally to this article.

Corresponding Authors: Xiao-Feng Zhu, Sun Yat-sen University Cancer Center,651 Dongfeng Road East, Guangzhou 510060, China. Phone: 8620-8734-3149;Fax: 8620-8734-3170; E-mail: zhuxfeng@mail.sysu.edu.cn; and Rong Deng,dengrong@sysucc.org.cn

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�2019 American Association for Cancer Research.

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blocking the IFNg-induced expression of immunosuppressivefactors may potentiate the therapeutic efficacy of ICB.

In melanoma, treatment strategies have mainly focused ontargeted therapy and ICB since 2011, when vemurafenib andipilimumab were approved by the FDA (13, 14). Although thesetreatments have drastically improved patient outcomes comparedwith conventional therapy, the targeted therapies using dabrafe-nib, vemurafenib, and trametinib are incapable of achieving long-lasting remissions due to chemoresistance, and the benefit fromICB therapy was observed only in a small fraction of patients(15, 16). The combination of ICB with conventional or targetedtherapies has become the center of cancer treatment (17).

We performed flow cytometry–based screening to identifykinase inhibitors that can block the IFNg-induced PD-L1 andIDO1 expression in melanoma. Here, we found that the FDA-approved agent regorafenib is one of themost promising agents inenhancing antimelanoma immunity, alone or in combinationwith ICB, throughdecreasing the expression level of IFNg-inducedPD-L1 and IDO1 without compromising cell surface MHC-Ilevels.

Materials and MethodsCell culture

Melanoma cell lines MM200, ME4405, MEL-RM, and B16/F10were purchased from ATCC. Cells were maintained in DMEMsupplemented with 10% FBS and antibiotics (50 mg/mL peni-cillin/streptomycin). All cell lines were verified to ensure theywere free of mycoplasma contamination before performing anyexperiments. Cell lines were authenticated by STR profilingthrough the Victorian Centre for Functional Genomics (mostrecent authentication, March 1, 2017).

MiceIn this study, C57BL/6 and BALB/C-nu/nu nude mice were

purchased from the Laboratory Animal Center of Guangdongprovince (Guangzhou, China). About 6-week-oldmalemice wereused for the experiments. All animal experiments were conductedin accordance with the institutional guidelines and approved by

the Animal Care and Use Committee of Sun Yat-sen UniversityCancer Center (Guangzhou, China).

Clinical dataData of 26 patients with advanced melanoma were retrieved

from cbioPortal (http://www.cbioportal.org/study?id¼skcm_ucla_2016#summary). All patients were treated with either pem-brolizumab or nivolumab as the anti–PD-1 therapy for theirmetastatic melanoma. Objective response to anti–PD-1 therapywas based on investigator-assessed immune-related ResponseEvaluation Criteria in Solid Tumors (irRECIST; refs. 18, 19). Dataof 163 patients with stage III melanomawithout immunotherapywere download from the The Cancer Genome Atlas (TCGA)database. Data of eight TH-MYCN mice bearing detectable spon-taneous neuroblastomas treated with anti–PD-1/L1 or controlwere downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/gds/).

Antibodies and reagentsThe antibodies used for Western blotting are listed in Supple-

mentary Table S1A. The secondary antibodies of goat anti-mouse(1:5,000) and goat anti-rabbit (1:5,000) were purchased fromThermo Fisher Scientific. The kinase inhibitor library, the inhibi-tors, and cytokines are provided in Supplementary Table S1B.

All siRNAs were obtained from GenePharma and transfectionwas performed using Lipofectamine RNAiMAX TransfectionReagent (Life Technologies, 13778-150) according to the manu-facturer's protocol. Information for primer sequences is providedin Supplementary Table S1C.

Flow cytometry analysisMelanoma cells were harvested and centrifuged at 400 � g (or

1,500 rpm) for 5 minutes. For cell surface staining, cell suspen-sions were washed twice in PBS and stained with indicatedfluorescent labeled antibodies for 30 minutes on ice and washedwith PBS. For intracellular staining, the cells were sorted forfixation and permeabilization using the Cytofix/CytoPerm BUFKIT (BD Pharmingen and; no. 554714). Immune cell infiltrationof tumors was analyzed as described previously (20). Briefly,tumors were resected from mice on indicated time, weighed,mechanically disrupted to generate a single-cell suspension andstained. All flow cytometry analyses were conducted on LSRFor-tessa (BDBiosciences) or aNavios andGallios (BeckmanCoulter)and the data were analyzed using FlowJo and CytExpert softwareaccording to the manufacturers' instructions. Compensationbeads were used to evaluate spectral overlap, and compensationwas automatically calculated. All antibodies used for flow cyto-metry analysis are listed in Supplementary Table S1D.

qRT-PCRqRT-PCR was conducted as described previously (21). cDNA

was reversed from 1 mg of total RNA using the QuantiTect ReverseTranscription Kit (Qiagen). SYBR Master Mix Plus (Eurogentec)was used for qPCR. Real-time PCR was conducted with a Bio-RadCFX96 qPCR system. Expression of GAPDH gene in each samplewas used as an internal control. Both forward and reverse primersare listed in Supplementary Table S1E.

Western blottingWestern blotting was carried out as described previously (22).

Whole-cell extracts were prepared by direct lysis with 1�Cell LysisBuffer (Cell Signaling Technology, no. 9873) and 1 mmol/L

Translational Relevance

Understanding the regulation of feedback expression of PD-L1, IDO1, and MHC-I in the IFNg abundant tumor microen-vironment is the key to relieving ICB resistance. This studyreported that regorafenib, an oral multi-kinase inhibitor usedto treat patientswith advanced gastrointestinal stromal tumorsand metastatic colorectal cancer, can block the IFNg-inducedPD-L1 and IDO1 expressionwhilemaintainingMHC-I expres-sion in melanoma. Hence, ICB-induced antitumor immuneresponse can be potentiated by the combination of regorafe-nib. Our study further demonstrated that the RET–Src axis canregulate both JAK1/2–STAT1 and MAPK signaling pathways.Altogether, our findings highlighted that regorafenib is apromising inhibitor in improving the antitumor immunity,and these findings might pave the way for a new therapeuticparadigm in treating patients with melanoma, especially withRET/Src axis activation.

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phenylmethylsulphonyl fluoride immediately before use. Allprotein samples were denatured with 6� SDS buffer and boiledat 100�C for 10 minutes and then, resolved by SDS-PAGE.

Cytotoxicity assays in vitroHuman peripheral blood mononuclear cells (PBMC) were

isolated from leukopheresis by Ficoll–Hypaque density gradi-ent. Isolated human PBMCs were subjected for CD3þ T-cellisolation by using the Pan T Cell Isolation Kit (Miltenyi Biotec)as instructed in the manual. OT-1 T cells were isolated fromspleen of OT-I TCR transgenic mice using the MojoSortTMMouse CD8 T Cell Isolation Kit (BioLegend # 480007) accord-ing to the manufacturer's protocol.The 12-well plates werecoated with LEAF Purified- anti-CD3 (10 mg/mL) and LEAFPurified anti-CD28 (2 mg/mL) in PBS overnight at 4�C or for2 hours at 37�C and washed with ice-cold PBS twice. Theisolated T cells were then plated into the 12-well plates toactivate for 72 hours in RPMI medium containing 10% FBS andhuman IL2 (100 IU/mL).

Melanoma cells were treated with indicated inhibitors with orwithout IFNg exposure for 24hours orfirst transfectedwith siRNAfor 48hours and then stimulatedwithorwithout IFNg for another24 hours.The prepared melanoma cells and activated T cells wereseeded into 96-well round bottom plates. The cells were sortedand applied for flow cytometric analysis.

Cell proliferation, migration, and invasion assaysCell viabilities were assessed with 3-(4, 5-dimethylthiazole-2-

yl)-2, 5 biphenyl-2H-tetrazolium bromide (MTT) assay. Thespectrophotometric absorbance at 570 nm was detected for eachsample, and the experiments were performed in triplicate andrepeated for three times. For colony formation assay, cells wereseeded in a 6-well plate and cultured for 2 weeks in completeDMEM medium containing indicated agents with or withoutIFNg exposure. Colonies were fixed and dyed with 0.1% crystalviolet (1 mg/mL), and the number of colonies with over 50 cellswas counted. Cell invasion was evaluated using a Transwell assaywith matrigel (8-mm pore; BD Biosciences). Melanoma cellswere seeded at a density of 0.5 � 105 into the upper chamber in200 mL DMEM medium without FBS, but containing indicatedagents with or without IFNg exposure. Complete medium (600mL) was added to the bottom chamber (i.e., 24-well transwellplate where the transwell inserts were added). After incubationfor 24 hours, the migratory cells were harvested and stainedwith 0.1% crystal violet (1 mg/mL). Wound healing assays wereperformed to detect cell migration. The cells were seeded in6-well plates, and an artificial wound was created using a 200mL pipette tip. The wound closure was observed after 24 hours,and images were obtained.

Animal experimentsAn inoculum of 0.5 � 106 B16 cells was injected subcutane-

ously on the flank of mice in 100 mL sterile PBS. Seven daysfollowing injection, mice were randomized into four groups andtreatment was initiated. The anti–PD-1 antibody (100 mg/mice)was administered intraperitoneally, regorafenib (3 mg/kg/dayin Fig. 5, 5 mg/kg in Fig. 4) was administered intragastrically,and IFNg (2.5 mg/mice) was administered by injecting directlyinto the tumor mass. Tumor volume was measured every otherday using the formula (L � W2/2).

Statistical analysisFor Gene Set Enrichment Analysis (GSEA), the JavaGSEADesk-

top Application was downloaded from http://software.broadinstitute.org/gsea/index.jsp. Statistical analyses were conductedusing GraphPad Prism (version 5.0, www.graphpad.com) andSPSS version (SPSS, Inc.). Student t test was used to compareexperimental data. Kaplan–Meier plots of overall survival (OS)and progression-free survival (PFS) were compared using the log-rank test. All data are presented asmean� SD, otherwise noted inthe figure legends. All reported P values are two-tailed, and for allanalyses, P < 0.05 is considered statistically significant, unlessotherwise specified. All data shown are representative of two ormore independent experiments, unless indicated otherwise.

ResultsRegorafenib is a potent agent attenuating IFNg-induced PD-L1and IDO1 expression

To identify a novel small-molecule kinase inhibitor that couldsuppress IFNg-induced cell surface PD-L1 expression, we per-formed flow cytometry–based screening using a kinase inhibitorlibrary (purchased from Selleck) composed of 429 compoundstargeting major 52 kinases. A collection of 161 agents was tested,making sure that each kinase had more than one inhibitor.Following stimulation with IFNg , we added the inhibitors at twodifferent concentrations (1 and10mmol/L) to the culturemediumof the melanoma cell line MEL-RM. After 24 hours of incubation,we detected PD-L1 expression by flow cytometry analysis(Fig. 1A). We found approximately 20 agents that caused morethan half reduction of cell surface PD-L1 level. Most of these hitswere JAK inhibitors, CDK inhibitors, MAPK inhibitors, mTORinhibitors, and the multi-kinase inhibitor regorafenib (Fig. 1B;Supplementary Table S2A). In addition, IFNg provoked MHC-Iexpression and enhanced the antigen presentation. Subsequently,we further examinedwhether these inhibitors had an influence onMHC-I expression. As presented in Fig. 1C and SupplementaryTable S2B, regorafenib was the only agent that inhibited PD-L1expression without reducing cell surface MHC-I levels.

To validate the results mentioned above, two other melanomacell lines, MM200 (BRAF V600E) and ME4405 (BRAF WT), weretreated with different concentrations of regorafenib in the pres-ence or absence of IFNg for 24 hours. The results revealed thatregorafenib inhibited the IFNg-induced PD-L1 and p-STAT1 andthe constitutive p-ERK1/2 in a dose-dependent manner (Fig. 1D;Supplementary Fig. S1A). Aberrant activation of the p-ERK,p-STAT1, and p-AKT were the main signaling events to drivePD-L1 expression, while regorafenib could not inhibit p-AKT.Hence, we next investigated whether inhibiting p-ERK or p-STAT1was sufficient to attenuate IFNg-induced PD-L1 expression. Ourresults indicated that regorafenib suppressed both the IFNg-induced p-STAT1 and the constitutive p-ERK, similar to the threeMAPK inhibitors (Fig. 1E; Supplementary Fig. S1G). Because onlyregorafenib reduced the IFNg-induced PD-L1 expression, asassessed by Western blot analysis, flow cytometry, and RT-PCR(Fig. 1E–G; Supplementary Fig. S1B–S1D, S1G–S1K), we con-cluded that inhibiting the p-STAT1 but not p-ERKwas sufficient toattenuate IFNg-induced PD-L1 expression. In addition, IFNg is apotent cytokine to induce IDO1 expression; therefore, we alsoevaluated and determined that only regorafenib inhibited theIFNg induced IDO1 expression depending on suppressingp-STAT1 (Fig. 1E and H; Supplementary Fig. S1B, S1E, S1G,

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S1L, and S1M). As a previous study indicated that MAPK pathwaydecreased MHC-I expression (23), whereas p-STAT1 promotedMHC-I expression, we further tested the cell surface MHC-I level.As displayed in Fig. 1I and Supplementary Fig. S1F, S1N, and S1O,vemurafenib significantly reduced the IFNg-induced MHC-I level

whereas regorafenib, dabrafenib, and trametinib had mild effects.Collectively, these data showed that regorafenib was a potent agentin reducing adaptive PD-L1 and IDO1 expression withoutcompromising the antigen presentation (maintainingMHC-I level)through repressing the JAK1/2–STAT1pathwayandMAPKpathway.

Figure 1.

Kinase inhibitor screening identified the potent inhibitor regorafenib blocking IFNg induced PD-L1 and IDO1 expression. A, Schematic of the screening strategy.One hundred thousand MEL-RM cells were seeded in the 12-well plates overnight. Following IFNg (125 IU/mL) stimulation, the inhibitors were added to the culturemedium at different concentrations (1 or 10 mmol/L). After 24 hours, cells were harvested and labeled with fluorescence conjugated anti–PD-L1 or anti–MHC-Iantibody. Fluorescence intensity was captured by flow cytometry. Statistical analysis was performed to identify agents for secondary screening and finalselection of agents. B, Results of the kinase inhibitor screening. Mean fluorescence intensity (MFI) fold change¼ [(inhibitorþ IFNg) MFI – isotype MFI]/(IFNg MFI– isotype MFI). MFI change values of PD-L1 for each agent were plotted to identify agents with fold change <0.5 at 10 mmol/L. Red filled circles represent hitssatisfying these thresholds. C, Results of the secondary screening. MFI change values of MHC-I for each agent identified from Bwere plotted to identify hits withfold change >0.8 at 10 mmol/L. Red filled circle represents regorafenib.D, Treatment with different concentrations of regorafenib in ME4405 cells for 24 hoursdecreased the P-ERK, P-STAT1, and PD-L1 levels. E–I, The data were obtained fromME4405 cells treated with indicated inhibitors (10 mmol/L) with or withoutIFNg (1000 IU/mL) exposure for 24 hours. E, Changes in STATs, ERK, and Akt pathway activity and PD-L1 and IDO1 expression levels. F,MFI fold change values ofPD-L1. G, Changes of PD-L1 mRNA level. H, Changes of IDO1 mRNA level. I,MFI fold change values of MHC-I. Error bars indicate SEM. Unpaired two-tailed t test,NS, not significant; ��� , P < 0.001; �� , P < 0.01; � , P < 0.05.

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RET–Src axis is required for regorafenib to inhibit JAK1/2–STAT1 signaling

Regorafenib is a multi-kinase inhibitor that blocks the activityof the oncogenic tyrosine kinases KIT, RET, and BRAF. Ourprevious data indicated that BRAF did not regulate JAK1/2–STAT1signaling (Fig. 1E); therefore, we further validated the targetbetween KIT and RET. Knockdown of RET but not KIT causedsuppression of p-JAK1, p-JAK2, p-STAT1, PD-L1, and IDO1 withIFNg exposure (Fig. 2A–C; Supplementary Fig. S2A), suggestingthat RET mediated the effect of regorafenib on JAK1/2–STAT1signaling. To exclude the confounding possibility that regorafenib

directly targeted JAK1/2, we added the inhibitor to the RETknockdown cells mentioned above, excluding the influence ofRET on regulating JAK1/2.Upon silencing of RET, regorafenib hadweak effect on further decreasing the p-JAK1, p-JAK2, p-STAT1,PD-L1, and IDO1 level (Fig. 2B). On the basis of the aboveobservations, we concluded that regorafenib indirectly sup-pressed the JAK1/2–STAT1 signaling through inhibiting the tyro-sine kinase receptor RET.

We next evaluated the contribution of more proximal RET-activated signaling enzymes to JAK1/2–STAT1 activation. Src isaberrantly activated in many tumors and was known to interact

Figure 2.

Regorafenib inhibited the JAK1/2-STAT1 pathway through RET–Src signaling. A and B, Effect of KIT or RET knockdown on JAK1/2-STAT1 pathway activity andPD-L1 expression. ME4405 cells were transfected with indicated siRNA for 48 hours and treated with regorafenib and IFNg for another 24 hours. C,MFI foldchange values of PD-L1 in RET knockdownME4405 cells with or without IFNg exposure. D, Decrease of p-Src in RET knockdownME4405 cells. E, Changes ofp-Src, p-ERK, and p-STATs activity in bosutinib-, PP2-, and regorafenib-treated ME4405 cells with or without IFNg exposure. F, Change of PD-L1 MFI values inbosutinib- and PP2-treated ME4405 cells with or without IFNg exposure. G,MFI fold change values of MHC-I in RET knockdownME4405 cells with or withoutIFNg exposure. H,MFI fold change values of MHC-I in bosutinib- and PP2-treated ME4405 cells with or without IFNg exposure. Error bars, SEM. Unpaired two-tailed t test. NS, not significant; ��� , P < 0.001; �� , P < 0.01; � , P < 0.05.

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with transmembrane receptor tyrosine kinases at the cell mem-brane via its SH2 and SH3 domains (24). Meanwhile, previousstudies also reported that Src could activate the JAK and STATfamilies (25, 26). Accordingly, we speculated that Src mediatedRET-induced JAK1/2–STAT1 activation. To investigate this,ME4405 or MM200 cells were treated with different concentra-tions of the Src inhibitor bosutinib, Lck/Fyn inhibitor PP2, andregorafenib as a positive control. Consistent with our hypothesis,RET knockdown in ME4405 cells decreased the p-Src level(Fig. 2D), and regorafenib also inhibited p-Src (Fig. 2E). More-over, bosutinib suppressed the induced p-STAT1, PD-L1, andIDO1 expression levels, similar to regorafenib (Fig. 2E and F;Supplementary Fig. S2B). Finally, we detected the MHC-I leveland found that RET–Src signaling did not affect the MHC-I level(Fig. 2G and H; Supplementary Fig. S2C and S2D). Taken togeth-er, regorafenib blocked the IFNg induced PD-L1 and IDO1expression without influencing MHC-I expression through inhi-biting RET–Src signaling.

Regorafenib recovered sensitivity of IFNg-pretreatedmelanoma cells to T-cell killing in vitro

Wehypothesized that PD-L1was themajor targetmediating theantitumor effect of regorafenib in the presence of IFNg . To verifythis, we used coculture experiments with melanoma cells toevaluate the effects of PD-L1 depletion or suppression on T-cellresponses. Melanoma cell lines were incubated with IFNg prior tococulture to induce PD-L1 expression. Cocultured with activatedhuman peripheral blood T cells, PD-L1 knockdown and regor-afenib pretreatment enhanced the sensitivity ofmelanoma cells toT-cell killing. Upon PD-L1 knockdown, regorafenib had a slighteffect on improving T-cell killing (Fig. 3A). To extend thesefindings using antigen-specific T-cell killing, OT-I T cells recog-nizing ovalbumin (OVA)-restricted epitopes were activated andcocultured with OVA-positive B16F10 cells. The data showed thatregorafenib sensitized the B16F10 cells to antigen-specific T-cellkilling (Fig. 3B). Together, IFNg-induced PD-L1 expression medi-ated melanoma cells escaping from T-cell killing and regorafenibregained the sensitivity depending on attenuating PD-L1expression.

Regorafenib is an FDA-approved drug for advanced gastroin-testinal stromal tumors and metastatic colorectal cancer, whiledabrafenib, vemurafenib, and trametinib are approved for mel-anoma. Tounderstand the effects of these agents inmelanoma,wefirst compared whether they affected the T-cell response. IFNg-pretreated cells were strongly resistant to T-cell killing, and onlyregorafenib-pretreated cells recovered the sensitivity to T-cellkilling (Fig. 3C and D; Supplementary Fig. S3A–S3D). As RET isthe target of regorafenib, we then cocultured the RET knockdowncells with activated T cells. Similarly, RET knockdown also poten-tiated T-cell killing (Fig. 3E and F).

We next validated whether regorafenib exhibited the sameantimelanoma functions as the MAPK inhibitors. Melanomacell lines were treated with various concentrations of dabrafe-nib, regorafenib, and vemurafenib in the presence or absence ofIFNg , and cells were assessed using MTT assay (SupplementaryFig. S3E and S3F), wound healing assay (Fig. 3G; Supplemen-tary Fig. S3G), soft agar colony formation assay (Fig. 3H;Supplementary Fig. S3H), and transwell assays (Fig. 3I; Sup-plementary Fig. S3I). These agents displayed similar abilities insuppressing proliferation, colony formation, migration, andinvasion. In conclusion, regorafenib is a promising antimela-

noma agent by two different mechanisms: a traditional mech-anism by targeting oncogenic addiction, and a newly definedmechanism through recovering sensitivity of IFNg-pretreatedmelanoma cells to T-cell killing.

Regorafenib enhanced the antitumor effect of IFNg in vivoNext, we wondered whether regorafenib exerted antitumor

potential in the IFNg-abundant tumor microenvironment invivo. To prove this point, we conducted tumor allograft andtreatment experiments using the melanoma cell line B16/F10.First, the cells were subcutaneously injected into C57BL/6 miceor nude mice. Following tumor formation, murine IFNg wasinjected into the tumor mass every 4 days to induce antitumorimmunity and stimulate PD-L1 and IDO1 expression. At thesame time, mice were treated with solvent or regorafenib everyother day. Mice were sacrificed 10 days later (Fig. 4A). InC57BL/6 mice with intact immunity, the combination ofregorafenib with IFNg led to a significant reduction in tumorgrowth and tumor weight (Fig. 4B and C). In nude mice withelimination of T cells, tumor growth curve and tumor weightfailed to show differences in each group (control, regorafenib,IFNg , and the combination groups; Fig. 4D and E). Collec-tively, we theorized that the tumor regression ability of regor-afenib combination with IFNg was dependent on intact T-cellimmunity.

Regorafenib enhanced the antitumor effect of ICB in vivoWeanalyzed the change in the IFNg pathway using 8 TH-MYCN

mice bearing detectable spontaneous neuroblastomas treatedwith or without anti–PD-1/L1 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE79485). The cellular response to IFNgand IFNg pathway genes, including PD-L1 and IDO1, was sig-nificantly increased (Supplementary Fig. S4A and S4B). Thisobservation suggested that anti–PD-1 therapy upregulated PD-L1 and IDO1 expression through the activation of the IFNgpathway in tumor cells.

We then hypothesized that regorafenib could enhance effectof ICB through blocking the feedback expression of PD-L1 andIDO1. The C57BL/6 mice bearing measurable melanoma xeno-grafts were randomly treated with solvent/isotype, regorafenib,anti–PD-1 antibody, or the combination (Fig. 5A). Regorafenibalone caused a 45% tumor reduction, and anti–PD-1 caused aslight tumor repression without statistical significance, which issimilar to Chen and colleagues' report (27). The combinedtreatment significantly improved the tumor growth inhibitionas confirmed by the growth curve of the xenograft tumorvolume and the tumor weight (Fig. 5B–D). Moreover, regor-afenib treatment eliminated the anti–PD-1 induced PD-L1 andIDO1 expression (Fig. 5E), but showed no obvious influence onMHC-I expression (Fig. 5F). To identify immune cell popula-tion changes, we analyzed immune cells infiltrating intotumors. CD4þT and CD8þT infiltration was relatively high(30%–40%) and similarly in each group (Fig. 5G–I). Combi-nation therapy stimulated more cytotoxic CD8þ T cells in theirtumor-infiltrating lymphocytes than those by each agent alone(Fig. 5F, J, and K). No obvious toxicity was observed in the micereceiving the dosage treatment. These findings suggested thatovercoming the resistance caused by feedback expression ofPD-L1 and IDO1, regorafenib was required for reinforcing theeffect of ICB.

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Figure 3.

Function of regorafenib or RET knockdown in melanoma cells. A, The percentage of cleaved caspase-3–positive cells among PD-L1 knockdown or regorafenib-pretreated ME4405 cells in the presence of IFNg . The cells were cocultured with activated human peripheral blood T cells for 8 hours. PD-L1 was effectivelyinterfered. B, The percentage of cleaved caspase-3–positive cells among regorafenib-pretreated B16/F10 cells in the presence of IFNg . The cells were coculturedwith activated OT-1 T cells for 3 hours. C and D, The percentage of cleaved caspase-3–positive cells among dabrafenib-, regorafenib-, and vemurafenib-pretreated ME4405 cells with or without IFNg exposure. B, Histogram of cleaved caspase-3 from a representative experiment. C, Summary of data from B. Graphshows fold change of cleaved caspase-3 when comparing the IFNg absence and presence groups. E and F, The percentage of cleaved caspase-3–positive cellsamong RET knockdown ME4405 cells with or without IFNg exposure. G, Cell migration of ME4405 cells after indicated treatment was measured by woundhealing assay (24 hours).H, Colony formation of ME4405 cells after indicated treatment was measured by clonogenic assay (10 days). I, Cell invasion ofME4405 cells after indicated treatment was measured by Transwell assay (16 hours). Error bars, SEM. Unpaired two-tailed t test. NS, not significant;��� , P < 0.001; �� , P < 0.01; � , P < 0.05.

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Patients with high coexpression of RET and Src showedunfavorable clinical outcome to anti–PD-1 treatment

Our data showed that inhibiting RET–Src signaling had aremarkable effect on blocking the feedback expression of PD-L1 and IDO1, prompting tumor T-cell infiltration and antitumorimmunity. We speculated that the patients with high RET and Srccoexpression in melanoma might be resistant to PD-1 blockadeimmunotherapy. To determine the clinical relevance, publiclyavailable data of 26 patients with melanoma who underwenttumor biopsies before starting immunotherapy (pembrolizumaband nivolumab) were obtained from the GEO datasets. Respond-ing pretreatment tumors (n¼ 13)were derived frompatients whohad complete or partial responses or stable disease control (withmixed responses excluded) in response to anti–PD-1 therapy.Nonresponding tumors (n¼ 13) were derived from patients whohad progressive disease. These response patterns were based onirRECIST. By interrogating their RNA-seq transcript with clinicalsignature, 10 patients with RET and Src co-high expression wereclassified as "High," while the 16 patients were categorized as"Other." A total of 80% patients were nonresponders in the"High" group, and only 31% patients were nonresponders in the"Other" group. This result was irrelevant to BRAF and NRASmutation state (Fig. 6A). Furthermore, we evaluated the influenceof RET and Src expression status on clinical benefits of anti–PD-1

immunotherapy as reflected by patient survival. Notably, the"Other" group was markedly associated with long OS and PFS(Fig. 6B and C; Supplementary Fig. S5A) compared with the"High" group. The univariate and multivariate Cox regressionanalyses indicated that RET and Src co-high expression was anindependent prognostic factor associatedwithpoorOSandPFS inanti–PD-1 treated patients (Supplementary Table S2C–S2E).Likewise, analysis of TCGA datasets of 163 patients with stageIII melanoma without any treatment also yielded similar results(Supplementary Fig. S5B and S5C; Supplementary Table S2F andS2G), but the survival difference was not as significant as thepatients treated with ICB. Moreover, immune response–relatedGSEA signatures were enriched in the "Other" group (Fig. 6D).Altogether, RET and Src co-high expression was an independentunfavorable prognostic factor through inhibiting the antitumorimmune response in melanoma.

DiscussionICB has resulted in long-lasting tumor regression, but the

response rate is relatively low. Hence, thorough understandingof the resistance mechanism is needed to improve the therapeuticefficacy. In this study, we reveal that the RET–Src axis has aprofound impact on antimelanoma immunity and is an

Figure 4.

Regorafenib enhanced theantitumor effect of IFNg dependingon intact T-cell immunity in vivo. A,In vivo experimental layout. B16/F10cells were inoculated into C57BL/6mice or nudemice. B and C,Xenograft volume and weight fromC57BL/6 mice (control and IFNg ,n¼ 6; regorafenib and combination,n¼ 6).D and E, Xenograft volumeand weight from nude mice (controland IFNg , n¼ 6; regorafenib andcombination, n¼ 6). Error bars,SEM. Unpaired two-tailed t test.NS, not significant; ��� , P < 0.001;�� , P < 0.01; � , P < 0.05.

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Figure 5.

Regorafenib enhanced the antitumor effect of anti–PD-1 by triggering CD8þ T-cell activation in vivo. A–D, Tumor growth of B16 cells in C57BL/6 mice(n ¼ 6/group). A, The treatment protocol is summarized by the arrows. B, Tumor volumes were calculated. C and D, Tumor weights from experimenton autopsy on day 15. E, Representative images of IHC staining of Gzmb, PD-L1, and IDO1 in B16 tumor sections after treatment. F, Quantitativeanalysis of MHC-I expression of each group as indicated. mRNA was obtained from the xenografts. G–I, Intracellular cytokine staining of CD8þ T-cellpopulations from isolated tumor-infiltrating lymphocytes. G, Representative dot plots of CD8þT in the TILs isolated from a representative mouse foreach group. H and I, Percentage of CD4þ T (H) and CD8þ T (I) in the TILs for each group. J and K, Intracellular cytokine staining of IFNgþ inCD45þCD8þ T-cell populations from isolated tumor-infiltrating lymphocytes. J, Representative dot plots of IFNgþCD8þ T in the TILs isolated from arepresentative mouse for each group. K, Percentage of IFNgþCD8þT in the TILs for each group. Error bars, SEM. Unpaired two-tailed t test. NS, notsignificant; ��� , P < 0.001; �� , P < 0.01; �, P < 0.05.

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independent unfavorable prognosis factor in melanoma patientswith or without ICB. Inhibition of the RET–Src axis by regorafenibblocks IFNg-induced feedback expression of PD-L1 and IDO1,reactivating cytotoxic CD8þ T cells in vitro and in vivo, andenhances the effect of IFNg or ICB in the immunocompetentmice. Mechanistically, we linked the RET–Src axis to the JAK1/2–STAT1 pathway. Thus, by targeting RET–Src axis, regorafenibovercomes the resistance mechanism and may increase the num-ber of patients who benefit from ICB (Fig. 6E).

IFNg is the most potent driver to induce PDL1 and IDO1expression, indicating that IFNg have immunosuppressiveroles leading acquired resistance to ICB (1, 3, 28). Here, weperformed flow cytometry–based screening to identify agents thatcan attenuate the IFNg-induced PD-L1 expression but withoutinfluencingMHC-I.We found regorafenibwas the agent satisfyingthis criterion, while JAK inhibitors, CDK inhibitors, MAPK inhi-bitors, and mTOR inhibitors blocked both PD-L1 and MHC-Iexpression.Much effort has beenmade to block the IFNg-induced

PD-L1 expression: loss of CMTM4/6 (29, 30) and CNS5 (31)decreased the PD-L1 protein pool through inducing the ubiqui-tination-mediated degradation, and CDK5 deletion suppressedPD-L1 transcription (9) while CDK4 kinase inhibitors stabilizedPD-L1 protein levels (32), indicating that these were promisingtargets. Because high levels of IDO1 upon IFNg stimulationstrongly inhibited the proliferation of effector T cells and stim-ulated the activation of Treg cells andmyeloid-derived suppressorcells (MDSC; ref. 12), much more attention should be paid tosimultaneously blocking PD-L1 and IDO1 expression. We deter-mined that regorafenib suppressed the IFNg-induced PD-L1 andIDO1 expression depending on inhibition of the RET–Src axis/JAK1/2–STAT1 pathway.

Besides, IFNg pathways have emerged as positive regulators ofresponse to ICB (33, 34). By inducing efficient antigen processingthrough upregulating MHC-I molecules, IFNg appears to be criti-cal for T-cell priming and activation (28, 35). Inhibiting JAK1/2–STAT1 signaling blocked MHC-I expression while suppressing

Figure 6.

The correlation between RET-Src expression and clinical response to PD-1 blockade therapy. A, Histogram representation of clinical benefit of anti–PD-1 based onRET and Src expression. Columns are shaded to indicate RET and Src expression status (high RET and Src coexpression group, red; the other group, green.Gender: M¼male; F¼ female. Vital status: A¼ alive; D¼ dead. Mutation status: WT¼wild; Mut¼mutation). Immune-related Response Evaluation Criteria inSolid Tumors (irRECIST): CR¼ complete response; PR¼ partial response; PD¼ progression of disease. B and C, Kaplan–Meier survival curves of OS and PFScompared between the high RET and Src coexpression group and the other group. The transcriptome data were obtained from patients with melanoma beforeanti–PD-1 therapy (GSE78220).D, GSEA between the high RET and Src coexpression group and the other group in patients before anti–PD-1 therapy(GSE78220). E, RET-Src co-high expression lead to superactivation of the JAK1/2–STAT1 pathway. By targeting the RET–Src axis and BRAF, regorafenib inhibitedthe MAPK and JAK1/2–STAT1 at the same time and subsequently attenuated the IFNg-induced PD-L1 and IDO1 expression without affecting MHC-I much.

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the MAPK pathway increased MHC-I expression (36). Throughinhibiting both the RET–Src axis/JAK1/2–STAT1 andMAPK path-way, regorafenib had little influence on MHC-I expression. Takentogether, through blocking the IFNg-induced PD-L1 and IDO1expression with little influence onMHC-I expression, regorafenibunleashed the IFNg side effect without compromising the antigenpresentation processes.

IFNg does regulate several aspects of the immune response.However, our results revealed that regorafenib indirectly inhibitedthe downstream JAK1/2–STAT1 pathway via the RET–Src axis inmelanoma cells. We found that RET deletion and Src inhibitionblocked IFNg-induced PD-L1 and IDO1 expressionwithout influ-encing MHC-I expression, recovering IFNg pretreatment melano-ma cells' sensitivity to T-cell killing. RET-Src promoted the JAK1/2–STAT1 pathway, significantly enhancing PD-L1 and IDO1expression. In patients treated with anti–PD-1 therapy, high RETand Src coexpression inhibited antitumor immune response andpredicted worse survival. This was consistent with the observa-tions that RET favored an immunosuppressivemicroenvironmentwith compromised CD8þ T-cell immunity (37). In the MT/retmodel of spontaneous metastatic melanoma, significantly highernumbers of Treg cells were recruited to skin tumors andmetastaticlymph nodes, while efficient Treg depletion did not delay mel-anoma development (38), indicating that inhibiting RET activitymight decrease infiltration of Treg cells and some other immu-nosuppressive cells and factors in the tumor microenvironment.In addition, some reports revealed that RET recruited tumor-infiltrating monocytic MDSCs and stimulated relatively highexpression of PD-L1, B7-H3, and B7-H4 coinhibitory moleculesin MDSCs (39, 40). Together with the previous studies, our studyindicated that theRET–Src axiswas amechanism for the resistanceto ICB and allowed for designing combination strategies toimprove the efficacy of ICB.

Next, we explored the influence of the treatment on CD8þ Tcells function in vivo. Our animal model showed the CD8þTinfiltration was relatively high (30%–40%) in each group.Anti—PD-1 or regorafenib alone did not increase CD8þT-cellinfiltration. It might be due to high levels of CD8þT-cell infiltra-tion basically in B16 mice model. The combination therapypromoted cytotoxic CD8þT-cell infiltration and caused obviouslytumor regression, while the function of regorafenib or anti–PD-1treatment alone was mild. It has been reported that overexpres-sion of PD-L1 induced by IFNg could limit the effect of anti–PD-1or PD-L1 antibodies (41). In fact, we observed inducible PD-L1expression after anti–PD-1 treatment in our animal models andGEO database. Therefore, the combinational therapy of regora-fenib and anti–PD-1 enhanced antitumor effect. In addition,anti–PD-1 treatment also caused inducible IDO1 expression. HighIDO1 activity leading to Trp depletion and Kyn accumulation inthe tumor microenvironment inhibited T-cell functions and stim-ulated Treg cells and MDSCs (12). This might also explain howmelanomas escape immune destruction despite ICB treatments. Inthis regard, regorafenib promoted cytotoxic CD8þ T-cell infiltra-tion and enhanced the efficacy of IFNg or ICB through decreasingthe feedback PD-L1 and IDO1 expression in vivo.

Patients with melanoma treated with MAPK inhibitors such asdabrafenib, vemurafenib, and trametinib rapidly develop drugresistance and recurrence after a short period of remission (15,16). Blocking the PD-L1/PD-1 inhibitory pathway reactivates theantitumor immune response and results in an objective and long-

lasting response without recurrence (42). Efforts have been madeto study whetherMAPK inhibitors have a role in regulating PD-L1expression. However, the observed results were ambiguous andcontradictory, as MAPK inhibitors have been reported to eitherinhibit PD-L1 or promote PD-L1 expression in the absence orpresence of IFNg exposure (43). Our results suggested that MAPKinhibitors had modest effects on PD-L1 and IDO1 expression inthe context of IFNg exposure and anti–PD-1 treatment. However,regorafenib was the most potent agent blocking IFNg-inducedPD-L1 and IDO1 expression. These results strongly suggested thatregorafenib was the optimal targeted drug in melanoma treat-ment, especially combined with ICB.

Our findings manifested a new model in patients with mela-noma with high RET and Src coexpression and had significantimplications for combined therapies. Regorafenib showed potentefficacy in promoting antitumor immunity through blocking theIFNg-induced expression of immunosuppressive factors, while ithad a mild effect on antigen processing, providing a mechanismfor new combinations of immunotherapies and targeted therapy.Ononehand, as our data showed, the combination of regorafeniband ICB significantly eradicates tumors. On the other hand, it is ofvalue to explore the effect of the combination of regorafenib withimmune stimulation therapy such as CAR-T or vaccine therapy inmelanoma, especially with RET/Src axis activation.

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

Authors' ContributionsConception and design: R.-Y. Wu, P.-F. Kong, L.-P. Xia, Y.-Y. Tang, R. Deng,X.-F. ZhuDevelopment of methodology: R.-Y. Wu, P.-F. Kong, Y. Huang, Z.-L. Li,Y.-Y. Tang, Y.-H. Chen, X. Li, H.-L. Zhang, Y. Yu, X.-D. Peng, L.-H. Zhou,G.-K. Feng, R. DengAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.-Y. Wu, P.-F. Kong, L.-P. Xia, Y. Huang, Z.-L. Li,Y.-Y. Tang, Y.-H. Chen, X. Li, H.-L. Zhang, Y. Yu, X.-D. Peng, L.-H. Zhou,G.-K. FengAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.-Y. Wu, P.-F. Kong, Y. Huang, Z.-L. Li, Y.-Y. Tang,Y.-H. Chen, X. Li, X.-L. Xu, Y. Yu, X.-D. Peng, L.-H. Zhou, R. DengWriting, review, and/or revision of the manuscript: R.-Y. Wu, P.-F. Kong,R. Senthilkumar, R. Deng, X.-F. Zhu, Y. Huang, Z.-L. Li, Y. ChenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.-P. Xia, T. Sun, X.-L. Xu, Y. Yu, J. Mai, D. Yang,R. Deng, X.-F. ZhuStudy supervision:R.-Y.Wu, P.-F. Kong, L.-P. Xia, Y.Huang, Z.-L. Li, Y.-H. Chen,X. Li, Y. Yu, X.-D. Peng, L.-H. Zhou, G.-K. Feng, R. Deng

AcknowledgmentsThis study was supported by the National Key R&D Program of China

(2017YFC0908501), the Natural Science Foundation of China (81630079,81772624, 81572605, 81572732, 81803006, 81802789), the Science andTechnology Project of Guangzhou (201803010007), the Natural Science Foun-dation of Guangdong Province (2017A030313481), and the FundamentalResearch Funds for the Central Universities of China (17ykjc25).

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 7, 2018; revised January 26, 2019; accepted March 26,2019; published first April 2, 2019.

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