TFEB Mediates Immune Evasion and Resistance to Mtor Inhibition of Renal Cell Carcinoma Via Induction of PD-L1

Published OnlineFirst August 5, 2019; DOI: 10.1158/1078-0432.CCR-19-0733

Translational Cancer Mechanisms and Therapy Clinical Cancer Research TFEB Mediates Immune Evasion and Resistance to mTOR Inhibition of Renal Cell Carcinoma via Induction of PD-L1 Cai Zhang1, Yaqi Duan2,3, Minghui Xia1, Yuting Dong2,3, Yufei Chen1, Lu Zheng4, Shuaishuai Chai5, Qian Zhang2,3, Zhengping Wei1, Na Liu1, Jing Wang1, Chaoyang Sun6, Zhaohui Tang7, Xiang Cheng8, Jie Wu9, Guoping Wang2,3, Fang Zheng1, Arian Laurence10, Bing Li5, and Xiang-Ping Yang1

Abstract

Purpose: Despite the FDA approval of mTOR inhibitors Results: TFEB did not affect tumor cell proliferation, (mTORi) for the treatment of renal cell carcinoma (RCC), the apoptosis, and migration. We found a positive correlation benefits are relatively modest and the few responders usually betweenTFEBandPD-L1expressioninRCCtumor develop resistance. We investigated whether the resistance to tissues, primary tumor cells, and RCC cells. TFEB bound mTORi is due to upregulation of PD-L1 and the underlying to PD-L1 promoter in RCCs and inhibition of mTOR molecular mechanism. led to enhanced TFEB nuclear translocation and PD-L1 Experimental Design: The effects of transcription factor EB expression. Simultaneous inhibition of mTOR and þ (TFEB) on RCC proliferation, apoptosis, and migration were blockade of PD-L1 enhanced CD8 cytolytic function evaluated. Correlation of TFEB with PD-L1 expression, as well and tumor suppression in a xenografted mouse model as effects of mTOR inhibition on TFEB and PD-L1 expression, of RCC. was assessed in human primary clear cell RCCs. The regulation Conclusions: These data revealed that TFEB mediates of TFEB on PD-L1 was assessed by chromatin immunopre- resistance to mTOR inhibition via induction of PD-L1 in cipitation and luciferase reporter assay. The therapeutic effi- human primary RCC tumors, RCC cells, and murine xeno- cacies of mTORi plus PD-L1 blockade were evaluated in a graft model. Our data provide a strong rationale to target þ mouse model. The function of tumor-infiltrating CD8 T cells mTOR and PD-L1 jointly as a novel immunotherapeutic was analyzed by flow cytometry. approach for RCC treatment.

Introduction Renal cell carcinoma (RCC) encompasses a heterogeneous group of cancers derived from renal tubular epithelial cells (1). Patients with localized RCC after partial or radical nephrectomy 1Department of Immunology, School of Basic Medicine, Tongji Medical College, often go on to develop metastatic disease, which requires systemic Huazhong University of Science and Technology (HUST), Wuhan, China. therapies that are rarely curative (2, 3). The mTOR is a serine/ 2Department of Pathology, School of Basic Medicine, Tongji Medical College, HUST, Wuhan, China. 3Institute of Pathology, Tongji Hospital, Tongji Medical threonine kinase that forms two complexes mTORC1 and College, HUST, Wuhan, China. 4Department of Neurobiology, School of Basic mTORC2 (4, 5). These sense the availability of nutrition, growth Medicine, Tongji Medical College, HUST, Wuhan, China. 5Department of Urology, factors, and energy levels to regulate cell growth, proliferation, Union Hospital, Tongji Medical College, HUST, Wuhan, China. 6Department of and differentiation. Dysregulation of mTOR pathways and muta- Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, HUST, tions of mTOR pathway–related genes are often associated with 7 Wuhan, China. Department of Surgery, Tongji Hospital, HUST, Wuhan, China. tumor growth in a variety of cancers (6, 7). Inhibitors of mTOR, 8Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College, HUST, Wuhan, China. 9Department of Cardio- everolimus, and temsirolimus that are derived from rapamycin vascular Surgery, Union Hospital, Tongji Medical College, HUST, Wuhan, China. have been approved by the FDA to treat advanced metastatic renal 10Department of Haematology, University College Hospital, London, England. cancers (8, 9). Despite initial excitement of mTOR inhibitors for Note: Supplementary data for this article are available at Clinical Cancer the treatment of RCC, mTOR inhibitors rarely achieve complete Research Online (http://clincancerres.aacrjournals.org/). responses and most patients ultimately develop resistance to mTOR inhibitor therapy (10, 11). However, the underlying C. Zhang and Y. Duan contributed equally to this article. mechanisms by which the RCC resists mTOR inhibition are Corresponding Author: Xiang-Ping Yang, Tongji Medical College, Huazhong elusive. University of Science and Technology, No.13, Hangkong Road, Wuhan 430030, The transcription factor EB (TFEB) is a member of the micro- China. Phone: 8627-8369-2600; Fax: 8627-8369-2608; E-mail: [email protected] phthalmia family of basic helix-loop-helix-leucine-zipper (bHLH-Zip) transcription factors (MiT family), which binds to Clin Cancer Res 2019;XX:XX–XX the coordinated lysosomal expression and regulation (CLEAR) doi: 10.1158/1078-0432.CCR-19-0733 consensus motif and plays important functions in regulation of 2019 American Association for Cancer Research. lysosome biogenesis, autophagy, and metabolism (12, 13). TFEB

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efficacy in a mouse RCC xenograft model. Thus, our data provide Translational Relevance evidence and rationale to support the combination of mTORi and Despite the significant progress achieved by targeting mTOR PD-L1 blockade as a potential therapeutic approach to treat RCC. in renal cell carcinoma (RCC), the effects of mTOR inhibitors are modest and patients often develop resistance. The lack of understanding of cancer cell–intrinsic mTOR-mediated path- Materials and Methods ways remains a major hurdle for the development of effective Cell culture therapies. Here, we uncovered that TFEB expression is posi- Cells were cultured at 37 C and 5% CO2 in a humidified tively correlated with PD-L1 expression in RCC cells. Further- incubator. 786-O, ACHN, H1975, A549, H2126, HCT116, LoVo, more, inhibition of mTOR in RCC enhances TFEB nuclear SW480, and Renca cells were from ATCC; 769-P, OS-RC-2, and localization and expression that subsequently drives PD-L1 Caki-1 cells were from and authenticated by Cell Repository, expression and immune evasion in RCC cell lines and primary Chinese Academy of Sciences (Shanghai, China). 786-O, ACHN, tumors. Simultaneously targeting mTOR and PD-L1 enhanced OS-RC-2, 769-P, and Caki-1 cells were authenticated by STR the efficacy in a mouse RCC xenograft model. Thus, our data profiling and authentication of other cell lines was not routinely provide rationale for a combinational strategy that targets performed. All of the cell lines were passaged less than 2 months mTOR and PD-1/PD-L1 axis jointly as a novel approach for after each thaw and tested to be free of mycoplasma contamina- patients with RCC. tion (D101, Vazyme).

Transient transfection and generation of stable cell lines 769-P, HEK 293T, and Renca cells were transfected with can be phosphorylated by mTORC1 and GSK3b at serine 211; either pcDNA3.1 control plasmid (EV) or pcDNA3.1 encoding serine phosphorylated TFEB interacts with 14-3-3 and is seques- the constitutively active form of TFEB S211A (TFEB) using tered within cytoplasm (14, 15). Mutation of serine 211 to lipofectamine 2000 (11668019, Invitrogen) as described by alanine (S211A) results in a loss of phosphorylation of TFEB by the manufacturer. TFEB mutant of TFEB-S211A was generated mTORC1 and leads to its constitutively nuclear localization (14). by site-directed point mutagenesis using MutanBEST kit (D401, TFEB can bind to the Tfeb promoter and induces its own expres- Takara). Transfected 769-P and Renca cells were selected with sion (16). The levels of the TFEB and its activity are elevated in G418 (A2513, APExBIO) for 3 weeks and subcloned by dilu- multiple types of human cancers and associated with proliferation at 1 cell/well in 96-well microliter plates. 786-O cells were tion and motility of cancer cells (17). Furthermore, TFEB fusion transduced with control lentivirus expressing a scrambled and overexpression caused by chromosomal translocation events shRNA or lentiviral particles encoding two TFEB short hairpin are linked with a poor prognosis in a subset of patients with RCC RNAs (shRNAs) as follows. ShTFEB-1 (forward): 50-CCGG- with elevated recurrence and metastasis (18, 19). CCCACTTTGGTGCTAATAGCTCTCGAGAGCTATTAGCACCAA- Cancer cells are subject to immune surveillance by which AGTGGGTTTTTG-30, ShTFEB-1 (reverse): 50-AATTCAAAAACC- cytotoxic T cells are able to recognize and kill tumor cells. RCC CACTTTGGTGCT AATAGCTCTCGAGAGCTATTAGCACCAAA- tumors are characterized by an inflammatory infiltrate and the GTGGG-30. ShTFEB-2 (forward): 50-CCGGCGATGTCCTTGGC- T-cell growth cytokine IL2 has long been used to treat RCC (20). TACATCAACTCGAGTTGATGTAGCCAAGGACATCGTTTTTG-30, To evade the immune system, tumor cells express coinhibitory ShTFEB-2 (reverse): 50-AATTCAAAAACGATGTCCTTGG CTAC- molecules, of which PD-L1 has attracted great interest (21). PD-L1 ATCAACTCGAGTTGATGTAGCCAAGGACATCG-30. Cells were expressed by the tumor cells binds to the inhibitory coreceptor selected with puromycin (HY-B1743, MCE) for 3 weeks and PD-1 on the surface of tumor-infiltrating T cells to suppress T-cell subcloned by dilution at 1 cell/well in 96-well microtiter plates. function. Blockade of the PD-L1/PD-1 axis has been of benefitin the treatment of many different types of cancers (21, 22). Cur- Immunoblotting rently, there are about 20 clinical trials for RCC at different stages Immunoblotting was performed according to standard method targeting PD-1, PD-L1, or both (23). with primary antibodies against TFEB (ab2636, Abcam), TFE3 It has been shown that PD-L1 expression in tumor cells is (SAB4503154, Sigma), PD-L1 (17952-1-AP, ProteinTech), regulated by many transcriptional factors including HIF-1a, GAPDH (5174, Cell Signaling Technology), PCNA (AV03018, STAT3, and NF-kB (24). Given the critical role of mTOR in the Sigma), BAX (2772, Cell Signaling Technology), phospho-S6 cross-regulation of tumor cells and functions of immune cells, in (4851, Cell Signaling Technology), phospho-4EBP1 (2855, Cell combination with the fact that TFEB is associated with RCC, we Signaling Technology), Histone H3 (17168-1-AP, ProteinTech), speculated that RCC might acquire the resistance to mTOR HIF-1a (14179S, Cell Signaling Technology), p65 (8242, Cell inhibition by upregulation of PD-L1 expression to promote Signaling Technology), phospho-p65 (3033, Cell Signaling Tech- immune evasion via TFEB. nology), STAT3 (12640, Cell Signaling Technology), and phos- In this study, we found that TFEB directly regulates PD-L1 pho-STAT3 (9145, Cell Signaling Technology), followed by incu- expression in RCC cell lines and primary human RCC cells, bation with appropriate secondary horseradish peroxidase– despite no effect on tumor cell biology. Overexpression of a conjugated antibodies, then evaluated with ECL (RPN2232, GE constitutively active form of TFEB (S211A) in RCC promotes Healthcare). tumor growth in immune competent but not nude mice by þ inhibiting antineoplastic CD8 T-cell function. Inhibition of Cell Counting Kit-8 and 5-ethynyl-20-deoxyuridine assay for cell mTOR enhances TFEB nuclear translocation and PD-L1 expres- proliferation sion in RCC cell lines and human primary renal cancers. Com- The different experimental groups of 786-O and 769-P cells bination of mTORi with anti-PD-L1 enhances the therapeutic were plated in 96-well plates at 1 103 cells per well and cultured

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for 5 days or 7 days, respectively. Cell proliferation was deter- with PE-conjugated antimouse PD-L1 antibody (124308, BioLe- mined by Cell Counting Kit-8 (CK04, Dojindo) every 24 hours gend) or FITC-conjugated antihuman/mouse Ki67 antibody (11- according to the manufacturer's instructions. The incorporation 5698-82, Invitrogen). The antibodies used to stain tumor- of 5-ethynyl-20-deoxyuridine (EdU) was stained with the EdU Cell infiltrating lymphocytes (TIL) were listed as followed: anti- Proliferation Assay Kit (Q10310-1, RiboBio) according to the CD45-APC/Cy7 (103115, BioLegend), anti-CD8-Percp/Cy5.5 þ manufacturer's protocol. The percentage of EdU for each field of (100734, BioLegend), anti-CD107a-APC (121613, BioLegend), view captured was recorded and quantified. anti-GZMB-FITC (515403, BioLegend), anti-IL2-BV421 (503825, BioLegend), anti-TNFa-PE/Cy7 (557644, BD), and anti-IFNg- Cell apoptosis determined by Annexin V-PI staining APC (554413, BD Biosciences). Samples were collected on a BD 786-O and 769-P cells were pretreated with DMSO or paclitaxel Verse Flow cytometer and data were analyzed using FlowJo (10 mmol/L) for 12 hours and then digested with 0.25% Trypsin software. (25200-114, Invitrogen) and washed twice with ice-cold PBS. Samples were stained with Annexin V-FITC (640906, BioLegend) Histology/IHC and propidium iodide (P4170, Sigma) in Annexin V binding RCC tissue specimens were isolated after surgery, formalin fixed buffer according to the manufacturer's specifications. Samples and paraffin embedded, and stained with hematoxylin and eosin. were analyzed on a BD Verse flow cytometer and data were IHC was performed as standard protocol with antibodies against analyzed using FlowJo software. TFEB (ab2636, Abcam), PD-L1 (clone 22c3, Dako), and carbonic anhydrase IX (CAIX) (TA336805, ZSGB-BIO, Beijing). Slides Transwell migration assay containing both PD-L1 positive and negative areas were taken The transwell migration assay was performed with standard for analysis of TFEB expression. Intensities of PD-L1 were deter- method. In brief, the bottom chambers were filled with 600 mLof mined according to the clinical score guideline for PD-L1 staining DMEM medium containing 10% FBS. In total, 2.5 104 cells were (clone 22c3, Dako). The 42 cases were divided into PD-L1 group suspended in 100 mL of DMEM containing 1% FBS and seeded in (<1%), PD-L1low group (1%–49%), and PD-L1high group the top chamber. After 24 hours, nonmigrated cells were removed (50%) with (1þ) PD-L1 cellular and membrane staining of and migrated cells were fixed with 4% paraformaldehyde and viable tumor cells. The tumor grades were assigned to the highest stained with 1% crystal violet. Images were taken using Olympus (5%) within the tumors if the tumors were heterogeneous. IHC model IX83 inverted fluorescence microscope and the percentages reactivity of cytoplasmic or nuclear TFEB was scored as follows: of migrated cell areas in the total areas were quantified. multiplication of the intensity of immunostaining (1, weak; 2, moderate; and 3, strong) and the percentage of positive tumor Mice cells, which resulted in a score of 0 to 300. The total TFEB Wild-type BALB/c and BALB/c nude mice (7–8 weeks of age, expression was evaluated as the mean of cytoplasmic and nuclear Huafukang) were housed in a specific-pathogen-free facility at the score. A score of less than 10 was considered as 0, a score of 10 to Tongji Medical College, HUST. All experiments were performed 40 was considered as 1þ, 41 to 140 as 2þ, and 141 to 300 as 3þ. according to the guidelines of the Institutional Animal Care and IHC data were evaluated by two independent pathologists. Use Committee of Tongji Medical College, HUST. Patients and specimens Xenograft mouse tumor models Studies with human RCC specimens have been approved by the In total, 1 106 Renca cells that were stably transfected with Ethics Committee of Tongji Hospital of HUST (Wuhan, China), either an empty vector or TFEB-S211A pcDNA3.1 plasmids were and signed informed consents were obtained from all patients' injected subcutaneously on the back of WT BALB/c mice. For the family. The demographic and clinical characteristics of the experiments performed with nude mice, 5 105 (EV or TFEB) enrolled patients are presented in Supplementary Table S1. Renca cells were injected. For the combinational therapy, WT BALB/c mice were subcutaneously injected with 1 106 Renca Chromatin immunoprecipitation assay cells. Once tumor volumes reached 50 to 100 mm3, mice were Cells were harvested followed by cross-linking for 10 minutes injected intraperitoneally with either 10 mg/kg temsirolimus with 1% (vol/vol) formaldehyde. Afterward, cells were lysed by daily, 200-mg anti-mouse PD-L1 antibody (10F.9G2, BioXCell) sonication. The cell lysates were immunoprecipitated with anti- every 3 days, combination of both, or vehicle together with TFEB (ab2636, Abcam) overnight at 4C. After washing and control rat IgG2b (LTF-2, BioXCell). Tumor volumes were mea- elution, cross-links were reversed for 4 hours at 65C. The eluted sured along major axis (a) and minor axis (b) daily and were DNA was purified and analyzed by qPCR using a Bio-Rad SYBR calculated using the formula: V ¼ ab2/2. Mice were sacrificed and Green intercalating fluorophore system with PD-L1 primers: tumors were excised and weighted. (forward): 50-AGTTTATGTGGC TGTGGGCA-30; PD-L1 primers: 0 0 (reverse): 5 -GGATATTTGCTGTCTTTATATTC -3 . The Ct value of Flow cytometry each sample was normalized to corresponding input value. For the analysis of PD-L1 and PD-L2 expression in human tumor cell lines or primary RCC cells, cells were harvested under Luciferase reporter assay normal condition or after administration of rapamycin (A8167, The PD-L1 promoter sequence (281 bp to þ43 bp) relative to APExBIO), Torin-1 (A8312, APExBIO), and EBSS (E2888, Sigma) the transcription start site was amplified by PCR from human for the indicated times. Cells were stained with antibodies against peripheral blood mononuclear cells and inserted into the pGL3- phycoerythrin (PE)-conjugated anti-human PD-L1 antibody basic vector (E1751, Promega). The primers used for cloning the (329706, BioLegend) and APC-conjugated antihuman PD-L2 PD-L1 promoter are: forward: 50-CGGCTAGCTGGGCAGATTT- antibody (345507, BioLegend). Mouse RCC cells were stained TTTTC-30 and reverse: 50-ATCTCGAGG CAAATGCCAGTAGG-30.

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HEK 293T cell was cotransfected with pRL-TK (E2241, Promega), tent with the knockdown data, enhanced TFEB expression did not pGL3-PD-L1 or pGL3-basic, empty pcDNA3.1 vector, or TFEB- affect 769-P cell proliferation, apoptosis, and migration in vitro S211A pcDNA3.1 plasmids in 24-well plates with Lipofectamine (Supplementary Fig. S1A–S1E). 2000. After 48 hours, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (E1901, TFEB mediates immune evasion of renal carcinoma cells Promega) with microplate reader (Synergy H1, Bio-Tek) and the We next explored the function of TFEB in an in vivo mouse ratio of firefly/Renilla luciferase was determined. model of RCC. To this end, we first generated a mouse-derived RCC Renca cell line that overexpressed TFEB-S211A mutant Isolation of primary tumor cells and TILs (Fig. 2A). Then we hypodermically inoculated control Renca- Tumor specimens were gently minced into small pieces and EV cells or Renca-TFEB (S211A) cells to wild-type BALB/c mice. then digested with 6 mL PBS containing 50 mL 25 mg/mL colla- We found that mice that received cells overexpressing TFEB-S211A genase IV (17104019, Invitrogen) and 25 mL 10 mg/mL DNase I showed significantly greater tumor burdens compared with con- (10104159001, Roche) for 1 hour at 37C. Cell suspensions were trol group (Fig. 2B). Greater tumor burdens were associated with filtered twice and centrifuged at 1,500 rpm for 5 minutes. Tumor significantly reduced frequencies of CD107a, GZMB, IL2, and þ cells and TILs were enriched and harvested separately by Percoll TNFa-producing CD8 cytotoxic cells (CTL) within tumors from gradient (17-0891-01, GE Healthcare) following the manufac- mice that received Renca cells overexpressing TFEB (Fig. 2C–F), turer's protocol. compared with mice that received control cells. There was a similar reduction in the percentages of IFN g-expressing CTL in Immunofluorescence TFEB-S211A Renca tumors, but this was not significant (Supple- þ Primary RCC cells were seeded on glass slides and incubated mentary Fig. S2A). In contrast, the percentages of CD107a NK overnight for proper attachment. Cells were stimulated with cells and presence of tumor-associated macrophages and mye- vehicle or Torin-1 (500 nmol/L) for 3 hours and then washed loid-derived suppressive cells within TFEB-S211A Renca tumors three times with PBS and fixed with 4% paraformaldehyde for 30 were unaltered compared with control Renca tumors (data not minutes, permeabilized with 0.05% Triton X-100 for 30 minutes, shown). and blocked in 5% BSA for 1 hour. Cells were incubated with anti- To resolve our contrasting findings between in vitro and in vivo TFEB (ab2636, Abcam) and anti-PD-L1 (clone 22c3, Dako) experiments, we repeated the same experiment with BALB/c nude overnight at 4C. Secondary antibodies labeled with FITC or Cy3 mice, which lack T cells. In contrast with the experiments per- (Life Technologies or Jackson Laboratories) were added for 1 hour formed on wild-type BALB/c host animals, there was no signif- at room temperature, and DAPI was used for nuclear counter- icant difference in tumor growth (Supplementary Fig. S2B and þ þ staining for 10 minutes. Samples were imaged with an Leica TCS S2C). Moreover, the percentage of Annexin V as well as Ki-67 SP5 confocal microscope in 24 hours after mounting. tumor cells in TFEB-S211A groups isolated from nude mice was comparable to the control group (Supplementary Fig. S2D and Statistical analysis S2E), suggesting that the effect of TFEB is dependent on the Statistical analysis was performed using Prism (GraphPad, presence of tumor-infiltrating T cells rather than intrinsic differ- San Diego) software. Statistical significance was determined by ences within the cells themselves. Student t test or for variances by ANOVA. P values less than 0.05 This led us to investigate the effect of TFEB on expression of were considered significant. inhibitory coreceptor ligands. We found that forced expression of TFEB in Renca cells enhanced PD-L1 expression within tumor tissues (Fig. 2G and H). In addition, the PD-L1 expres- Results sion levels within tumors were positively associated with TFEB does not affect cell proliferation, apoptosis, and tumorweights(Fig.2I).Takentogether,TFEBmediated migratory potential of RCC cells immune evasion of RCC via suppressing the cytotoxic function þ TFEB expression has been linked with both occurrence and a of CD8 Tcells. poor prognosis in RCC (19). To determine whether TFEB affects the proliferative, apoptotic, and metastatic capacities TFEB positively regulates PD-L1 expression in RCCs of RCCs, we took the advantage of the differential expression We next evaluated whether TFEB correlated with PD-L1 of TFEB in human 786-O and 769-P RCC cell lines (Fig. 1A). expression in primary RCC cells from patients. Within indi- Knockdown of TFEB in 786-O cells did not affect expression of vidual tumors, PD-L1 staining showed heterogeneous expres- PCNA, a marker for cell proliferation, as well as proapoptotic sion, which can be readily differentiated into PD-L1 and þ þ protein BAX expression (Fig. 1B). Consistent with these, PD-L1 areas. The PD-L1 regions had higher expression and knockdownofTFEBexpressiondidnotaffectcellproliferation enhanced nuclear localizations of TFEB (Fig. 3A and B); the (Fig.1C).Theseresultswerefurtherconfirmed with EdU representative images of differential intensities of cytoplasmic staining (Fig. 1D and E). Next, we looked at cell survival; or nuclear TFEB staining were shown in Supplementary knockdown of TFEB in 786-O cells had no effect on apoptosis Fig. S3A. To explore possible link between PD-L1 expression in untreated cells or cells treated with paclitaxel (Fig. 1F and and tumor progression, we extended the study to a cohort of 42 G). Finally, using an in vitro transwell assay, we found that patients with clear cell renal cell carcinoma (ccRCC). On the downregulation of TFEB in 786-O cells had no impact on cell basis of the percentages of PD-L1 expression in viable tumor migration compared with cells transduced with scrambled cells, patients are divided into three groups: PD-L1 (<1%), shRNA lentivirus (Fig. 1H and I). PD-L1low (1%–49%), and PD-L1high groups (50%). PD-L1 To confirm these conclusions, we overexpressed a constitutive high-expression patients had higher tumor grades and more active form of TFEB mutant, TFEB-S211A, in 769-P cells. Consis- advanced tumor stages (Fig. 3C and D). Next, we compared

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Figure 1. TFEB does not affect RCC proliferation, apoptosis, and migratory potential. A, TFEB expression was determined in 786-O and 769-P cells by immunoblot analysis. B, 786-O cells were transduced with a scramble shRNA (con shRNA) and TFEB shRNA (shTFEB-1, shTFEB-2) lentiviral particles. Expression of TFEB, PCNA, and BAX was determined by immunoblot analysis, and significance of changes of band intensities was determined by one-way ANOVA. C, Cell proliferation of 786-O cells was detected with CCK8 assay. D and E, Representative images of EdU incorporation in 786-O cells (D) and the percentages of EdUþ cells were determined in four different areas (E). F, Cell apoptosis was determined by PI/Annexin V staining in untreated or paclitaxel (10 mM, 24 hours) treated 786-O cells. Representative histograms (F) and significance were determined by one-way ANOVA (G). H and I, Representative images (H)and quantitative numbers of invaded cells (I) across the membrane porous over 24 hours were determined. Mean SEM; P < 0.001; ns, not significant. All experiments were repeated three times except D for twice.

surface PD-L1 expression in a panel of human RCC cell lines. expression of TFE3, another member of MITF, was not associ- Although ACHN cells had the highest expression of PD-L1, ated with PD-L1 expression in RCC cell lines (Fig. 3F; Supple- which is followed by 786-O, Caki-1, and OS-RC-2 cells, 769-P mentary Fig. S3B). cells had the lowest surface PD-L1 expression (Fig. 3E). We Knockdown of TFEB expression led to reduced PD-L1 expres- obtained similar results for PD-L1 expression using immuno- sion in 786-O cells (Fig. 3G and H), which was accompanied blotting analysis; furthermore, expression of TFEB was posi- with reduced TFEB binding to the PD-L1 promoter (Fig. 3I). tively correlated with the levels of PD-L1 (Fig. 3F). In contrast, Conversely, overexpression of TFEB in the 769-P cells

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Figure 2. RCC cells overexpressing TFEB suppress cytotoxic CD8þ T-cell function. A, Renca cells were transfected with either an empty vector (EV) or TFEB-S211A (TFEB) pcDNA3.1 plasmids to generate stable cell lines. TFEB expression was determined by immunoblot analysis. B–I, A total of 1 106 Renca cells (EV or TFEB) were injected subcutaneously on the back of WT BALB/c mice. Tumor growths were shown and significances were determined by t test (B; n ¼ 9). C–F, At day 23, mice were sacrificed, TILs were isolated, and cell surface was stained with antibodies against CD8 and CD107a (C), followed with intracellular staining with antibodies against Granzyme B (D), IL-2 (E), and TNFa (F; n ¼ 5–9). G, IHC staining of TFEB and PD-L1 within tumor tissues. Representative images are shown. H, Single-cell suspensions were prepared from tumor tissues, PD-L1 expression was determined on gated CD45 cells, and significance of MFI was determined by t test (n ¼ 7). I, The correlation of tumor weights and PD-L1 staining scores was determined (mean SEM; P < 0.05; P < 0.01; ns, not significant).

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Figure 3. TFEB regulates PD-L1 expression in RCC cells. A, H&E and immunohistochemical (IHC) staining with TFEB and PD-L1 on human RCC tissues. Representative images are shown. Scale bar, 50 mm (n ¼ 11). B, Immunoreactive score (IRS) of nuclear and total TFEB in human PD-L1-negative and PD-L1- positive RCC tissues measured by IHC (n ¼ 11). The associations of tumor grade (C) or tumor stages (D) with PD-L1 expression in a cohort of 42 patients with ccRCC. PD-L1 expression in RCC cell lines was determined by flow cytometry (E) or immunoblot analysis (F). The correlation of TFEB and PD-L1 expression was plotted. 786-O cells stably expressing a control shRNA or TFEB shRNAs were analyzed for cell-surface PD-L1 expression (G) and significance of MFI changes was determined by one-way ANOVA (H). I, Chromatins from 786-O cells (con shRNA, TFEB shRNAs) were precipitated with anti-TFEB. The immunoprecipitated DNAs were amplified by qPCR with primers spanning the PD-L1 promoter region. J, HEK 293T cells were transfected with the pGL3 basic plasmids, or pGL3-PD-L1-Luc plasmids together with either empty or TFEB-S211A plasmids for 48 hours, then luciferase activities were determined. Mean SEM; P < 0.05; P < 0.01; P < 0.001; ns, not significant; H&E, hematoxylin and eosin; all experiments (E–J) were repeated for three times with similar results.

significantly enhanced TFEB binding to the PD-L1 promoter mTOR inhibition enhances PD-L1 expression via activation of and PD-L1 expression (Supplementary Fig. S3C and S3D). TFEB in RCC cells Furthermore, transient expression of TFEB significantly Given that TFEB is a major target of mTORC1 (14), we asked enhanced luciferase activity driven by the PD-L1 promoter whether inhibition of mTOR could lead to changed expression of (Fig.3J).Together,thesefindings demonstrate that TFEB direct- PD-L1. Inhibition of mTOR pathway, either directly by rapamycin ly binds to the PD-L1 promoter and positively regulates PD-L1 and Torin-1 or indirectly by cell starvation, significantly enhanced expression. TFEB expression and its nuclear accumulation in 786-O cells

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Figure 4. mTOR inhibition enhances PD-L1 expression via TFEB in RCC cells. 786-O (A and B) and 769-P cells (C and D) were treated with 100 nmol/L rapamycin for 6 hours or 500 nmol/L Torin-1 for 3 hours or starved for 6 hours. PD-L1, S6 and 4-EBP1 phosphorylation, nuclear TFEB, and total TFEB expression in 786-O cells (A) or 769-P cells (C) were determined by immunoblot analysis. Cell surface PD-L1 expression in 786-O cells (B) or 769-P cells (D) was determined by flow cytometry. E, Cell lysates of 786-O or 769-P cells were prepared and chromatins were fragmented with sonication and precipitated with anti-TFEB. The immunoprecipitated DNAs were amplified for the PD-L1 promoter. F, 786-O cells were transduced with scramble shRNA and TFEB shRNA lentiviral particles and then treated with rapamycin, Torin-1, and starvation as described in A. The amounts of TFEB, PD-L1, and S6 phosphorylation were determined by immunoblot (F). The significance of intensity changes of TFEB (G) and PD-L1 (H) was determined by two-way ANOVA. All experiments were repeated for three times (mean SEM; , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant).

(Fig. 4A). Furthermore, this was associated with enhanced PD-L1 groups (Fig. 4E). To further substantiate that PD-L1 induction by expression (Fig. 4A and B). HIF-1a, STAT3, and p65 have all been mTOR inhibition was via activation of TFEB, we knocked down implicated as regulators of PD-L1 expression. In our hands, TFEB expression by shRNA in 786-O cells and looked at the effect inhibition of mTOR did not affect the phosphorylation of STAT3 of mTOR inhibition on PD-L1 expression. Knockdown of TFEB and p65 but significantly reduced HIF-1a expression (Supple- abolished the enhanced PD-L1 expression upon inhibition of mentary Fig. S4A and S4B). We found similar results in 769-P cells mTOR by rapamycin, Torin-1, or starvation (Fig. 4F–H). (Fig. 4C and D; Supplementary Fig. S4C and S4D). The negative Together, these data demonstrate that inhibition of mTOR regulation of mTOR on PD-L1 seemed to be RCC specific, as specifically promoted PD-L1 expression in RCC via TFEB inhibition of mTOR resulted in reduced expression of PD-L1 in activation. lung carcinoma H1975 cells (Supplementary Fig. S4E) and no change of expression in A549 and H2126 lung carcinoma cells, as mTOR inhibition enhances PD-L1 expression via TFEB well as HCT116, LoVo, and SW480 colon cancer cells (Supple- activation in human primary RCC cells mentary Fig. S4E and S4F). Next, we asked if the inhibition of mTOR enhanced PD-L1 Torin-1 treatment enhanced TFEB binding to the PD-L1 pro- expression in human patients with renal cancer. To this end, we moter in both 786-O and 769-P cells, compared with control isolated primary renal cancer cells from freshly surgically removed

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Figure 5. TFEB mediates enhanced PD-L1 expression upon mTOR inhibition in human primary renal cancer cells. A, H&E and IHC staining with anti-CAIX antibody on human ccRCC tissues. B, Immunofluorescence staining with anti-CAIX antibody on human primary ccRCC cells. C, Isolated human primary ccRCC cells were treated without or with 500 nmol/L Torin-1 for 30, 60, and 90 minutes. TFEB expression in fractions of cytoplasm and nuclear was determined. Significance of TFEB intensity changes was determined by one-way ANOVA. D–F, Human primary RCC cells were untreated or treated with 100 nmol/L rapamycin for 6 hours, 500 nmol/L Torin-1 for 3 hours, or 6-hour starvation. The amounts of TFEB, PD-L1, and S6 phosphorylation were determined by immunoblot analysis and significance of PD-L1 changes was determined (D). Cell surface PD-L1 (E) and PD-L2 (F) expression was determined by flow cytometry. G, Immunofluorescence staining with anti-TFEB and anti-PD-L1 antibodies on primary RCC cells treated with vehicle and 500 nmol/L Torin-1 for 3 hours. The experiments were repeated for three times (mean SEM; , P < 0.05; P, < 0.01; , P < 0.001; ns, not significant).

tumor tissues. IHC staining showed that tumor tissues had a were almost all CAIX-positive, indicating the purity of primary CAIX positive staining, a transmembrane protein and marker for cells (Fig. 5B). Torin-1 induced rapid drop in the cytoplasmic ccRCC (Fig. 5A). The isolated primary ccRCC cells from patients concentrations of TFEB at 30 minutes, followed by a slower

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increase in the nuclear concentration of TFEB (Fig. 5C). In line PD-L1 expression via TFEB activation in human primary renal with this, prolonged mTOR inhibition led to enhanced PD-L1 cancer cells. expression measured by immunoblot analysis and flow cytometry (Fig. 5D and E). In contrast, PD-L2 expression was not affected by Anti-PD-L1 immunotherapy enhances the response to mTOR mTOR inhibition (Fig. 5F). These results were confirmed using inhibition in RCC immunofluorescence staining of TFEB and PD-L1 (Fig. 5G). We then asked whether combination of antibody against Together, these data demonstrate that mTOR inhibition induces PD-L1 could potentiate the efficacy of mTOR inhibition by

Figure 6. Simultaneously targeting PD-L1 significantly enhances mTORi efficacy in a mouse xenograft model. A–F, Wild-type BALB/c mice were subcutaneously injected with 1 106 Renca cells. Once tumor volumes reached 50 mm3, mice were treated with vehicle and IgG, temsirolimus (TEM; 10 mg/kg), anti-PD-L1 (200 mg/mouse), or a combination of TEM, and anti-PD-L1 for 12 days (n ¼ 7–9). Tumor volumes were recorded daily. Schematic representation of the treatment was shown (A). Comparison of Renca tumor growth in different groups (B) and the mice were necropsied at day 24 and tumors were shown (C). D, Phosphorylation of S6 within CD45- negative tumor cells was determined by flow cytometry. E, H&E and IHC staining of TFEB, PD-L1, and Ki67 within tumor tissues. F, TILs were isolated and stained with CD8, CD107a, and GZMB. Representative histograms shown on the left panel. Percentages of CD107aþCD8þ or GZMBþCD8þ are shown on the right panel (mean SEM; , P < 0.05; , P < 0.01; , P < 0.001; ns, not significant).

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Resistance to mTORi in RCC via TFEB-mediated PD-L1 Induction

temsirolimus on ccRCC growth in a xenograft mouse model. directly binds to the PD-L1 promoter. Of note, the induction of When tumor volume reached 50 mm3, mice were treated with PD-L1 in RCCs was irrespective of the VHL status, as the tested either temsirolimus (10 mg/kg, i.p.) daily, anti-PD-L1 (200 mg per RCCs contain both VHL-negative and VHL-positive cells (27). mouse, i.p.) four times over 12 days, combination of both, or Consistent with this, PD-L1 expression was enhanced concurrent vehicle plus control IgG (Fig. 6A). There was a reduction in tumor with enhanced TFEB expression, despite reduced HIF-1a expres- growth in mice treated with either anti-PD-L1 alone or temsir- sion in both 786-O and 769-P cells upon mTOR inhibition. olimus alone inhibited tumor growth compared with the control Furthermore, knockdown of TFEB in RCC rendered the cells less group, but this was not significant (Fig. 6B and C). In contrast, the responsive to PD-L1 induction upon mTOR inhibition, indicating combination of temsirolimus and anti-PD-L1 therapy resulted in a critical role of TFEB in regulation of PD-L1 expression in RCC a significant reduction in tumor size compared with all other cells. groups and complete disappearance of tumors in 3 mice after The induction of TFEB and PD-L1 by mTOR inhibition seems to 23 days (Fig. 6B and C). Temsirolimus suppressed mTORC1 be RCC specific, as PD-L1 expression in colon cancer and lung activation in tumor cells as measured by ribosome S6 phosphor- carcinoma cells was not enhanced by mTOR inhibitors. The ylation and enhanced TFEB and PD-L1 expression within tumor activity of TFEB is tightly regulated by protein phosphorylation tissues (Fig. 6D and E; Supplementary Fig. S5A), compared with at multiple serine sites, which can be mTOR-dependent (S122) or control group, indicating the in vivo significance of TFEB–PD-L1 -independent (S138 and S134; refs. 28, 29). It is conceivable that axis during tumor growth. Although temsirolimus did not sig- the activity of TFEB is independent of mTOR regulation in non- nificantly inhibit tumor cell proliferation assessed by Ki67 IHC RCCs. In line with this, the phosphorylation and nuclear trans- staining, anti-PD-L1 led to a small but significant reduction in location of TFEB can be regulated by GSK3b in breast cancer Ki67 staining and combination of PD-L1 and temsirolimus had a cells (15). A PARP inhibitor that inactivated GSK3b in breast synergistic effect on suppression of Ki67 staining (Fig. 6E; Sup- cancer cells can enhance PD-L1 expression and cancer-associated plementary Fig. S5B). immunosuppression (30). It is tempting to speculate that PARPi Next, we tested the effect of mTOR and PD-L1 inhibition on induces PD-L1 expression via activation of TFEB. þ cytotoxicity in tumor-infiltrating CD8 T cells. Temsirolimus In contrast to the positive role of PI3K-AKT-mTOR in the treatment suppressed CD107a and GZMB expression in CTL; regulation of PD-L1 expression in lung carcinoma cells (31), anti-PD-L1 treatment had little effect but the combination of mTOR inhibition led to enhanced PD-L1 expression in RCC cells anti-PD-L1 and temsirolimus significantly enhanced their expres- in our hands, which is consistent with a previous study (32). PI3K- sion (Fig. 6F). The combination of the two significantly enhanced AKT-mTOR can regulate PD-L1 expression following growth IFN g expression compared with the CTL from untreated animals, factors or inflammatory stimuli in both an IFN g-dependent and although IFNg was not inhibited by temsirolimus treatment alone -independent manner in non–small cell lung cancer (NSCLC), and was enhanced by anti-PD-L1 alone (Supplementary glioma, breast cancer, and melanoma cells (24). Together, these Fig. S5C). Together, these data demonstrate that only the com- data highlight the contextual roles of PI3K-AKT-mTOR in regu- bination of inhibiting mTOR and PD-L1 signaling significantly lation of PD-L1 expression in tumor cells. induced the expression of all three markers of T-cell cytotoxicity. Although checkpoint and mTOR inhibitors have been success- ful as cancer therapies, as monotherapies these drugs seem to be insufficient to fully block cancer progression (33, 34). Consistent Discussion with our findings, targeting mTOR and PD-1/PD-L1 axis simul- Identifying the mechanism by which tumors are resistant to taneously has improved efficacies in treatment of oral cavity targeted mTOR inhibitors has largely focused on analysis of cancer and hepatocellular carcinoma (35, 36). Although the intracellular signaling pathways with limited focus on the enhanced tumor control with combination of mTOR and PD- immune microenvironment of the tumors (25, 26). In this L1 targeting depends on CTL but not NK cells (35), some of the study, we demonstrated that inhibition of mTOR in RCC cell mechanisms may be different. mTOR inhibition leads to lines and human primary RCC cells leads to enhanced trans- enhanced MHC-I expression in oral cavity tumors; in HCC, location and expression of TFEB, which subsequently induces PD-1 promotes tumor growth via enhancing the phosphorylation PD-L1 expression. Furthermore, combination of mTOR inhi- of 4-EBP1 and ribosomal protein S6 (36). bition and anti-PD-L1 enhanced the cytotoxic functions of In summary, our data demonstrated that TFEB mediates PD-L1 tumor-infiltrating CTL and therapeutic efficacy in a mouse RCC upregulation by mTOR inhibitors, which can attenuate mTORi xenograft model. therapeutic efficacy via tumor-associated immune suppression. TFEB has been linked with many aspects of cellular events These data provide strong scientific rationale for the combination including proliferation, metabolism, and autophagy (12, 13). Yet of mTOR-targeted therapy and anti-PD-L1 immunotherapy, in our hands, alteration of TFEB expression has little intrinsic which may benefit patients with RCC. effects on in vitro RCC tumor biology including cell proliferation, survival, and migration. Only in the presence of T cells did we see Disclosure of Potential Conflicts of Interest the effect of TFEB. Identification of the TFEB target genes in RCC No potential conflicts of interest were disclosed. cells may provide better understanding of the functions of TFEB in regulation of RCC tumorigenesis and the interaction between Authors' Contributions RCC cells and the immune microenvironment. Conception and design: C. Zhang, Y. Duan, J. Wu, X-P. Yang Development of methodology: C. Zhang, Y. Duan, M. Xia, Y. Dong, Y. Chen, PD-L1 expression in cancer cells is regulated by a variety of L. Zheng, X-P. Yang transcriptional factors including HIF-1a, NF-kB, STAT1, and Acquisition of data (provided animals, acquired and managed patients, STAT3 (24). Our studies revealed a strong association between provided facilities etc.): S. Chai, Q. Zhang, Z. Wei, N. Liu, J. Wang, C. Sun, PD-L1 protein and TFEB expression in RCC cells, in which TFEB Z. Tang, F. Zheng, G. Wang, B. Li

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Analysis and interpretation of data (e.g. statistical analysis, biostatistics, (to Z. Tang), and Integrated Innovative Team for Major Human Diseases computational analysis): C. Zhang, Y. Duan, M. Xia, X-P. Yang Program of Tongji Medical College, HUST (to X-P. Yang). A. Laurence is Writing, review, and/or revision of the manuscript: C. Zhang, Y. Duan, M. Xia, supported by the Crohn's & Colitis Foundation of America. A. Laurence, X-P. Yang, X. Cheng Administrative, technical, or material support (i.e. reporting or organizing The costs of publication of this article were defrayed in part by the data, constructing databases): Y. Duan, S. Chai, G. Wang payment of page charges. This article must therefore be hereby marked Study supervision: X-P. Yang advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments This work was supported by grants from the National Scientiﬁc Foundation Received March 2, 2019; revised June 11, 2019; accepted August 1, 2019; of China (#31870892, #81671539, and #31470851, to X-P. Yang), 81873870 published ﬁrst August 5, 2019.

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TFEB Mediates Immune Evasion and Resistance to mTOR Inhibition of Renal Cell Carcinoma via Induction of PD-L1

Cai Zhang, Yaqi Duan, Minghui Xia, et al.

Clin Cancer Res Published OnlineFirst August 5, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-0733

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