Inhibition of Nr4a Receptors Enhances Anti-Tumor Immunity by Breaking Treg-Mediated Immune Tolerance

Author Manuscript Published OnlineFirst on March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-3102 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Inhibition of Nr4a receptors enhances anti-tumor immunity by breaking Treg-mediated immune tolerance

Sana Hibino1, Shunsuke Chikuma1, Taisuke Kondo1, Minako Ito1, Hiroko Nakatsukasa1, Setsuko Omata-Mise1, and Akihiko Yoshimura1,2

1Department of Microbiology and Immunology, Keio University School of Medicine 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

2Correspondence to: Akihiko Yoshimura, PhD Department of Microbiology and Immunology Keio University School of Medicine

35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Phone: +81-3-5363-3483 Fax: +81-3-5360-1508 Email: [email protected] Running title: Promotion of anti-tumor immunity by Nr4a inhibition Keywords: regulatory T cell, immune tolerance, tumor immunology, transcription

factors, animal models of cancer Conflict of Interest Statement: The authors declare no potential conflicts of interest. Acknowledgements: We thank N. Shiino, M. Ohkura, Y. Tokifuji, and Y. Hirata for their technical assistance. This work was supported by JSPS KAKENHI (S) 17H06175 (to A. Yoshimura), Advanced Research & Development Programs for Medical Innovation (AMED-CREST) JP17gm0510019 (to A. Yoshimura), the Takeda Science Foundation (to A. Yoshimura), the Uehara Memorial Foundation (to A. Yoshimura), the SENSHIN Medical Research Foundation (to A. Yoshimura), and Grant-in-Aid for Scientific Research on Innovative Areas 17H05801 (to S. Chikuma).

Downloaded from cancerres.aacrjournals.org on October 7, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on March 20, 2018; DOI: 10.1158/0008-5472.CAN-17-3102 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract Enhanced infiltration of regulatory T (Treg) cells into tumor tissue is detrimental to cancer patients and closely associated with poor prognosis as they create an

immunosuppressive state that suppresses anti-tumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg cell genetic programs, contribute to Treg-mediated suppression of anti-tumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin (CPT) and a common cyclooxygenase-2 (COX-2) inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted anti-tumor effects. Genetic inactivation or pharmacological inhibition of Nr4a factors unleashed effector activities of CD8+ cytotoxic T cells and evoked potent anti-tumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacological modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment.

Significance This study reveals the role of Nr4a transcription factors in Treg-mediated tolerance to anti-tumor immunity, with possible therapeutic implications for developing effective anticancer therapies.

Introduction CD4+CD25+ regulatory T (Treg) cells, characterized by transcription factor forkhead box P3 (Foxp3) expression, play critical roles in maintaining immunological self-tolerance and homeostasis (1,2). Tregs provide dominant regulation over self-reactive conventional T cells by inhibiting their expansion and activation (3). Tumor cells are derived from self-tissues, but they could be recognized as non-self and eradicated by the immune system because of the expression of tumor-specific mutated genes. However, tumor cells with fewer immunogenic mutations are close to “self”, and likely evade immune surveillance, resulting in tumor progression. In this case, mechanisms for maintaining self-tolerance, such as Tregs, may play undesired roles by establishing immune tolerance against tumors (4,5). Additionally, tumor tissue itself creates a niche that supports survival and function of Tregs (6). An increased abundance of Tregs and a decreased ratio of intratumoral CD8+ cytotoxic T cells (CTLs) to Tregs have been shown to predict a poor prognosis in various types of human cancers (7).

Tregs behave as major obstacles in clinical application of cancer immunotherapy, such as tumor vaccines or immune check point blockade (4,8). Therefore, breaking immunosuppression by Tregs is important for successful cancer therapy. Depletion of Tregs or specific disruption of Treg functions are actually thought to be promising strategies to enhance anti-tumor immunity (4,8,9). Chemical inhibitors targeting signal pathways or molecules that contribute to the suppression activities of Tregs have been intensively studied (10-13). For example, the master transcription factor Foxp3 is an attractive target, and P60, a peptide inhibitor of Foxp3, was reported to inhibit Treg function and improve tumor vaccine efficiency (14). However, there is

not enough evidence to show that Foxp3 alone is sufficient to be targeted for disruption of Treg function because multiple factors work cooperatively with Foxp3 to maintain Treg physiology (15,16).

We recently discovered that the Nr4a family of nuclear orphan receptors, consisting of three isoforms (Nr4a1, Nr4a2, and Nr4a3), redundantly play essential roles in Treg development and function via their ability to directly transactivate Foxp3 expression (17-19). Thymic Treg development was completely inhibited in mice lacking all three of the Nr4a factors on T cells (CD4-Cre Nr4a1fl/fl Nr4a2 fl/fl Nr4a3-/-), and they died within 3 weeks because of systemic multiorgan autoimmunity (18). In addition, Nr4a factors have been shown to be highly expressed on mature Foxp3+ Tregs and necessary for maintaining Treg stability and suppressive activities (19). Of note, Nr4a factors not only regulate Foxp3 expression but also globally regulate the Treg-specific transcriptional program. Therefore, Nr4a-deficient Tregs showed not only reduced expression of Foxp3, but also global dysregulation of Treg signature genes, including Foxp3-independent genes such as Ikzf4 (Eos). Thus, we hypothesize that Nr4a factors are a promising target for cancer immunotherapy to disrupt Treg function within tumors. In this study, using the mouse tumor transplantation model, we found that selective deletion of Nr4a1/Nr4a2 within Foxp3+ Tregs significantly suppressed tumor growth

and induced potent anti-tumor immune responses. We then searched for pharmacological modulators of Nr4a factors, and identified two well-known drugs: the classical chemotherapeutic agent camptothecin (CPT) as the inhibitor of Nr4a transcriptional activity and cyclooxygenase (COX)-2 inhibitors, such as the celecoxib analogue SC-236, as the inhibitor of Nr4a transactivation. These drugs synergistically exerted potent anti-tumor immune responses against mouse tumor models in an Nr4a/Treg-dependent manner. We propose that Nr4a factors play important roles in Treg-mediated suppression of anti-tumor immunity, and they are attractive therapeutic targets for cancer immunotherapy.

Methods Mice All experiments using mice were approved by the Institutional Animal Care and Use Committee (IACUC; approval number 08004) of Keio University, and performed according to IACUC guidelines. The Nr4a1- and Nr4a2-floxed mice, and Foxp3YFP-Cre knock-in mice were previously described (19). C57BL/6J mice were purchased from Tokyo Laboratory Animals Science (Tokyo, Japan). IFN-γ-venus reporter mice were reported previously (20). All mice were maintained on a C57BL/6 genetic background and kept in specific pathogen-free conditions at Keio University.

Cell lines and culture All cell lines were obtained between 2008-2014. Authenticated 3LL (Lewis lung carcinoma) tumor cells were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). MC38 (colon adenocarcinoma) tumor cells were kindly provided by Dr. James P. Allison. Human embryonic kidney 293T

cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA USA). 3LL cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MC38 and 293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were frozen down at early passages (<7) and used in the experiments within 5 passages after thawing. These cells were not further authenticated by our laboratory; however, routine confirmation of in vitro growth properties, morphology, tumor formation in the C57BL/6 syngeneic mouse strain (3LL,

MC38), and transfection efficiency (293T) provided evidence of correct cell identity. They were negative for known mouse pathogens, including mycoplasma.

Drugs (S)-(+)-Camptothecin and 7-Ethyl-10-hydroxycamptothecin (SN-38) were purchased from Tokyo Chemical Industry (Tokyo, Japan). Prostaglandin E2 (PGE2) and thymidine were purchased from Nacalai Tesque (Kyoto, Japan). Z-VAD-FMK was purchased from Peptide Institute (Osaka, Japan). CPT-11 (irinotecan HCl trihydrate) was purchased from Biochempartner (Shanghai, China). SC-236 was purchased from Sigma-Aldrich (St. Louis, MO, USA). H-89 was purchased from Cayman Chemical (Ann Arbor, MN, USA). Mitomycin-C was purchased from Kyowa Hakko Kirin (Tokyo, Japan). The Screening Committee of Anticancer Drugs (SCADS) inhibitor kits (http://gantoku-shien.jfcr.or.jp/) containing 363 compounds were kindly provided by a Grant-in-Aid for Scientific Research in the Priority Area “Cancer” from the Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan).

Fluorescence activated cell sorting (FACS) The following fluorescently labeled antibodies were purchased from BioLegend (San

Diego, CA, USA), eBioscience (San Diego, CA, USA), or TONBO biosciences (San Diego, CA, USA): CD4(RM4-5), CD8(53-6.7), CD25(PC61.5), CD3ε (145-2C11), CD11b(M1/70), CD11c(N418), CD45.1(A20), CD80(B7-1; 16-10A1), CD86(B7-2; GL1), Foxp3(FJK-16s), IFN-γ(XMG1.2), TNF-α(MP6-XT22), IL-4 (11B11), CD107a(1D4B), CD152(CTLA-4; UC10-4B9), Ki-67(16A8), and Nr4a1(12.14). Dead cells were gated-out using Fixable Viability Dye eFluor 780 (eBioscience). For intracellular cytokine staining, cells were stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich) in the presence of Brefeldin A (eBioscience) for 4 h. Cells were stained for surface antigens,

and then fixed/permeabilized with Foxp3 fixation/permeabilization concentration and diluent (eBioscience), and stained for nuclear proteins or cytokines. For phospho-CREB (pCREB) staining, cells were fixed/permeabilized with 4% paraformaldehyde and

ice-cold 90% methanol, and stained with pCREB (Ser133) (87G3) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA 01923, USA) for 1 h, followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Waltham, MA, USA). Stained samples were measured using a BD Canto II (BD Biosciences, San Jose, CA, USA) and FlowJo software (Tree Star, Ashland, OR, USA) was used for data analysis.

Mouse tumor models Before transplantation into mice, 3LL and MC38 tumor cells were grown to 80% confluence, then counted and suspended in PBS. Each mouse was subcutaneously injected with 2.5×105 3LL or 5.0×105 MC38 tumor cells into the right flank on day 0. Tumor growth was monitored every 3-4 days throughout the experiment. Tumor volume was calculated using the following formula: 0.5×ab2 (a, major axis; b, minor axis). For inhibitor treatment experiments, mice were injected intraperitoneally with CPT-11 (75 mg/kg) and/or the COX-2 inhibitor SC-236 (3 mg/kg) on days 6 and 9. For

CD8+ T cell depletion, mice received an intraperitoneal injection of 400 μg CD8+ T cell-depleting antibody (TIB211) three times per week beginning 6 days after tumor implantation.

Isolation of immune cells from tumor At the specified time points after tumor implantation and drug treatment, subcutaneous tumors were excised, minced, and digested with 1 mg/ml collagenase D (Roche, Basel, Switzerland) and 100 μg/ml DNase I (Roche) at 37°C for 1 h with gentle shaking. Cells were passed through a 70-μM filter, following low speed centrifugation (400 rpm, 5

min) to remove cell debris and epithelial cells. Red blood cell lysis was then performed if necessary, and cells were washed twice with PBS and used for further experiments.

Mouse T cell isolation Spleen and lymph node cell suspensions were prepared from the indicated mice, and CD4+ T cells were isolated using a mouse CD4+T Cell Isolation Kit and autoMACS Pro (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD62L+ naïve T cells were prepared as previously described (21). For preparation of CD4+CD25+ Tregs, isolated CD4+ T cells were stained with allophycocyanin (APC)-conjugated anti-mouse CD25 antibody, followed by positive selection with anti-APC microbeads using autoMACS. Over 95% of the sorted CD4+CD25+ cells expressed Foxp3. When Foxp3YFP-Cre knock-in mice were used, MACS-isolated CD4+ cells were stained with PerCP-cy5.5 anti-mouse CD4 antibody, and CD4+YFP+ cells were sorted using a SONY SH800 cell sorter (Sony, Tokyo, Japan).

Culture of primary T cells TCR (T cell receptor) stimulation was performed by incubating T cells with anti-CD28 Ab (57.31; 2μg /ml) and plate-coated anti-CD3ε Ab (145-2C11; 4 μg/ml). Th-skewing

conditions in this study were described previously (17). Retrovirus transduction to primary T cells was performed as described previously (16,21). All cultures were performed in RPMI1640 supplemented with 10% FBS, 1% penicillin/streptomycin, 100 nM non-essential amino acids, 2 mM glutamine, and 55 μM 2-mercaptoethanol (Invitrogen).

Adoptive transfer of Treg cells Isolated Tregs were activated using Dynabeads Mouse T-Activator CD3/CD28 (Thermo Fisher Scientific), according to the manufacturer’s instructions. At the specified time

points, 2.5×105 cells were transferred intravenously into the tumor-bearing host.

In vitro suppression assay

Responder T cells (CD45.1+CD4+CD25− cells) were prepared from C57BL/6J CD45.1 congenic mice, and labeled with 1 µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen). CFSE-labeled responder cells (5 × 104) were co-cultured with unlabeled CD45.1- Tregs at the indicated ratio in the presence of Dynabeads Mouse T-activator CD3/CD28 (Thermo Fisher Scientific) in round-bottomed 96-well dishes at a concentration of 1 bead per cell. The CFSE dilution of CD45.1+ responder cells was measured using flow cytometry 96 h later.

mRNA preparation and quantitative RT-PCR Total RNA was extracted using RNAiso Plus (Takara Bio, Shiga, Japan) or ReliaPrep RNA Cell Miniprep System (Promega, Madison, WI, USA), and subjected to reverse transcription (RT) using a High Capacity cDNA Synthesis Kit (Thermo Fisher Scientific). PCR analysis was performed using an iCycler iQ multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA) and SsoFast EvaGreen Supermix

(Bio-Rad). All primer sets yielded a single product of the correct size. Relative expression levels were normalized to 18S rRNA. Sequences for primers used in this study are available upon request.

Luciferase assay 293T cells were seeded on 24-well plates and transfected with pCMV expression plasmids and pGL4-luciferase plasmids, 200 ng each, using polyethylenimine. 24 h after transfection, the indicated chemicals were added to the culture and incubated for an additional 18 h. Cells were lysed in 100 μl lysis buffer, and luciferase activity was

measured using a luciferase substrate kit (Promega) and bioluminescence-monitoring apparatus (CL96; Churitsu). For each transfection, 200 ng β-galactosidase was added as an internal control. Luciferase assay-based drug screening was performed as described

in Supplementary methods.

Statistical analysis For comparison of two groups, statistical analysis was performed using a Student’s t-test or a Mann-Whitney U-test. ANOVA with Tukey's post hoc test was used for multiple comparison experiments. p-values <0.05 were considered to indicate statistical significance (*p< 0.05, **p<0.01, ***p<0.001).

Results Treg-specific deletion of Nr4a1/Nr4a2 restricts tumor growth Human effector Treg fraction (Foxp3hiCD45RA-CD25hi; fraction II) has been shown to be highly suppressive and dominant in tumor tissues, among the three fractions of human Tregs (9,22). Interestingly, we confirmed that Nr4a factors are most highly expressed in the fraction II population of human Tregs (Supplementary Fig. S1A,B). This suggests that Nr4a factors can be a target for modulating tumor-associated Tregs.

Then, to investigate the role of Nr4a/Tregs in anti-tumor immunity, we prepared

Treg-specific Nr4a1/Nr4a2 double-conditional knockout mice (Foxp3YFP-Cre Nr4a1fl/flNr4a2fl/flNr4a3+/+; hereafter called Nr4a-DcKO mice). We used Foxp3YFP-Cre Nr4a1+/+Nr4a2+/+ Nr4a3+/+ as the WT control. Unlike Nr4a-triple KO (Foxp3YFP-Cre Nr4a1fl/flNr4a2fl/flNr4a3-/-) mice (19), Nr4a-DcKO mice did not show any spontaneous inflammation, suggesting that systemic Nr4a3 sufficiency suppresses autoimmunity. Overall, the T cell population in the thymus and periphery, including the Treg cell fraction in CD4+ T cells, was normal in Nr4a-DcKO mice (Supplementary Fig.

S2A,B). However, Tregs from Nr4a-DcKO mice had significantly lower expression levels of Treg-signature genes, such as Foxp3, CD25, and CTLA-4, at both mRNA and protein levels (Supplementary Fig. S2C,D). We also confirmed that in vitro

suppression activity of Nr4a-DcKO Tregs was attenuated compared with WT (Supplementary Fig. S2E). Additionally, at steady state, IFN-γ+ cell fraction was increased within both CD4+ and CD8+ conventional T cells in Nr4a-DcKO mice (Supplementary Fig. S2F, upper and middle panels). We did not find any IL-4-producing CD4+ cells in Nr4a-DcKO mice, suggesting the absence of Th2 type inflammation that is characteristic in TKO mice (Supplementary Fig. S2F, bottom panels). To examine the effect of the Nr4a1/Nr4a2 deletion in Tregs on anti-tumor immunity, we performed a mouse subcutaneous tumor transplantation model using the syngeneic 3LL tumor cell line. Although tumor growth rates were indistinguishable between WT and Nr4a1 or Nr4a2 single cKO mice, Nr4a-DcKO mice showed a remarkable delay of tumor growth (Fig. 1A). The established tumor was almost completely eradicated in some Nr4a-DcKO mice, but not in WT mice (Fig. 1B). The tumor-resistant phenotype of Nr4a-DcKO mice was also confirmed when the MC38 (colon adenocarcinoma) cell line was transplanted (Fig. 1C,D,E).

Enhanced anti-tumor immunity in Nr4a-DcKO mice We then assessed the contribution of immune cells to the suppression of tumor growth observed in Nr4a-DcKO mice. As shown in Fig. 2A, transfer of in vitro activated WT Tregs into 3LL tumor-bearing Nr4a-DcKO mice almost completely abolished tumor growth inhibition, suggesting that delayed tumor growth was due to functional defects of Nr4a-DcKO Tregs. Additionally, antibody-mediated depletion of CD8+ CTLs in Nr4a-DcKO mice also accelerated tumor growth, suggesting that CD8+ CTLs are the main effector population that restricts tumor growth in Nr4a-DcKO mice (Fig. 2B).

Based on these findings, we further examined immune responses that occurred in 3LL tumor-bearing mice. In tumor-draining lymph nodes (TDLNs), the fraction of CD8+ T cells was significantly increased in Nr4a-DcKO mice compared with WT mice

(Fig. 2C), and cells expressing the proliferation marker Ki-67 were also increased among these CD8+ T cells, suggesting the active expansion of CD8+ CTLs in TDLNs (Fig. 2D). On the other hand, accumulation of Foxp3+ Tregs in TDLNs was inhibited in Nr4a-DcKO mice (Fig. 2E). Additionally, Foxp3 protein expression was also significantly down-regulated in TDLN Tregs of Nr4a-DcKO mice (Fig. 2F). In tumor tissue, enhanced T cell infiltration was observed in Nr4a-DcKO mice (Fig. 2G, upper panels). The fraction of intratumoral Foxp3+ Tregs was not affected (Fig. 2G, lower panels), but the CD8+/Treg ratio was significantly higher in Nr4a-DcKO mice compared with the WT mice (Fig. 2H). Additionally, the anti-tumor effector CD8+CTLs, characterized by the expression of effector cytokines (IFN-γ, TNF-α) and degranulation marker CD107a, as well as IFN-γ+Th1 CD4+T cells were drastically increased in Nr4a-DcKO mice (Fig. 2I), suggesting the induction of potent anti-tumor immune responses. Similar results were obtained from the experiments using MC38 tumor cells (Supplementary Fig. S3A,B,C). We also confirmed that enhanced immune responses in Nr4a-DcKO mice occurred in a tumor antigen-specific manner by examining in vitro

recall responses (Supplementary Fig. S3D). We then examined the mechanism of effector CTL enhancement by Nr4a-DcKO Tregs. Previous research demonstrated that intratumoral Tregs suppress co-stimulatory signals from antigen-presenting cells (APCs) by CTLA-4-mediated trans-endocytosis of B7-molecules CD80/CD86, which in turn suppress CD8+CTL effector function (3,23-25). We observed significant up-regulation of CD80 expression on tumor-infiltrating dendritic cells (DCs), as well as down-regulation of CTLA-4 on tumor-infiltrating Tregs, in Nr4a-DcKO mice (Supplementary Fig. S3E,F,G). Collectively, these results strongly support our hypothesis that inhibition of Nr4a factors

in Tregs breaks immune tolerance against tumor cells and facilitates anti-tumor activities of tumor-infiltrating effector CTLs.

Identification of camptothecin as a functional Nr4a inhibitor Crystal structure analysis revealed that Nr4a factors function as ligand-independent transcription factors, and their pharmacological antagonists have not been identified (26). Therefore, we conducted reporter assay-based drug screening to identify functional Nr4a inhibitors from a chemical library (Methods summarized in Supplementary Fig. S4A). We looked for compounds that inhibited the transcriptional activity of Nr4a2, and identified camptothecin (CPT) as a potent Nr4a inhibitor (An overview and results of the screening are described in detail in Supplementary Fig. S4B,C). CPT is a DNA topoisomerase I (topo I) inhibitor that was shown to be effective in a broad spectrum of tumors (27). CPT inhibited not only Nr4a2, but also Nr4a1 and decreased Nr4a response element (NBRE) reporter activity in a dose-dependent manner (Fig. 3A). CPT also suppressed Nr4a2-induced transactivation of Foxp3-promoter reporter activity (Fig. 3B). While the CPT analogue topotecan inhibited Nr4a activity similarly to CPT, other topoisomerase inhibitors, such as daunorubicin or etoposide, did not, suggesting that suppression of Nr4a by CPT is independent of inhibition of

topoisomerase activity (Supplementary Fig. S4D). Additionally, CPT did not affect the transcriptional activity of other nuclear receptors such as estrogen receptor α (ERα), thyroid-hormone receptor β (Thrβ), or peroxisome proliferator-activated receptor γ (PPARγ) (Supplementary Fig. S4E), revealing that CPT is not a general transcription inhibitor, but is specific to Nr4a. To further confirm the screening result, we next examined the effect of CPT on mouse primary T cells. CPT treatment suppressed Foxp3 protein expression induced by ectopic expression of Nr4a2 in naïve CD4+ T cells (Fig. 3C), demonstrating that CPT also inhibits Nr4a2 transcriptional activity in T cells. In addition, under in vitro helper T

cell differentiation conditions, CPT potently suppressed induction of Foxp3+ induced Treg (iTreg) cells while it promoted induction of IFN-γ+ Th1 cells in a TGF-β-independent manner, at both mRNA and protein levels (Fig. 3D,

Supplementary Fig. S5A,B). Since deletion of Nr4a in T cells robustly suppress iTreg differentiation while promoting Th1 differentiation (17), these results indicate that CPT treatment mimicked the effects of Nr4a-knockdown in T cells. Similar results were obtained in the presence of the apoptosis inhibitor Z-VAD-fmk, suggesting that these effects are not a result of cytotoxic effects of CPT (Supplementary Fig. S5C). We then examined the effect of CPT on the stability of Tregs in vitro. We treated CD4+CD25+ Tregs (>95% Foxp3+ Treg) with CPT in the presence of thymidine and Z-VAD-fmk to inhibit cell proliferation and apoptosis, respectively. CPT significantly reduced the expression of Foxp3 and other Treg-related genes in a dose-dependent manner (Fig. 3E,F). CPT-induced destabilization of Foxp3 expression was not observed in Tregs from Nr4a-DcKO mice, suggesting that CPT reduces Foxp3 through the inhibition of Nr4a factors (Fig. 3G). On the other hand, the expression of Nr4a factors themselves were not affected by CPT treatment (Fig. 3E), which is consistent with our proposal that CPT inhibits transcriptional activity of Nr4a factors, but not their expression.

In vivo effects of CPT on Foxp3+ Tregs were also examined using CPT-11 (irinotecan), a water-soluble, less toxic prodrug of CPT (28). Since CPT-11 exerted its effect after being metabolized into SN-38 (7-Ethyl-10-hydroxy-camptothecin) in vivo, we confirmed that SN-38 inhibited Nr4a activity in vitro similarly to CPT (Supplementary Fig. S5D,E). We also confirmed that any significant side effects, such as weight loss or immunosuppression were not observed in naive mice treated with the dose of CPT-11 used in this study (Supplementary Fig. S5F,G). Although CPT-11 treatment did not affect the population of T cell subsets including Tregs in the thymus and periphery (Supplementary Fig. S5H,I), the Foxp3 expression levels in Tregs were

significantly decreased (Fig. 3H). These data indicate that CPT destabilizes Foxp3 expression both in vitro and in vivo. Taken together, in addition to the well-defined chemotherapeutic effects, our data revealed a previously unidentified inhibitory activity

of CPT on Nr4a function.

COX-2 inhibitor targets the PGE2-Nr4a axis on Treg cells in tumor microenvironment COX-2 is often constitutively overexpressed in a variety of tumors, and its enzymatic product PGE2 contributes to tumor progression either by direct effects on tumor cells or through the formation of a tumor-promoting microenvironment (29). Interestingly, in intestinal tumors, PGE2 has been shown to induce Nr4a2, thereby supporting survival and proliferation of tumor cells, and COX-2 inhibitors abolished such effects (30,31). Therefore, we investigated the effect of PGE2 and COX-2 inhibitors on Nr4a1/Nr4a2 expression in Tregs. Transient stimulation with PGE2 alone for 3 h upregulated both Nr4a1 and Nr4a2 expression in Tregs in a dose-dependent manner (Fig. 4A,B). Additionally, continuous PGE2 stimulation for 18 h also enhanced Nr4a1/Nr4a2 expression in Tregs that were maintained by the TCR stimulation (Fig. 4C). Induction of Nr4a expression by PGE2 was dependent on cAMP/PKA (protein kinase A), because

PKA inhibitor H-89 abolished these effects (Fig. 4B,C). Of note, PGE2 also enhanced expression of Foxp3 and Ikzf4 (Eos), direct targets of Nr4a, in WT Tregs, but not in Nr4a-DcKO Tregs (Fig. 4D). These data indicate that PGE2 promotes the expression of Nr4a factors and regulates Nr4a-dependent gene expression in Tregs. Both 3LL and MC38 tumor cells constitutively express COX-2 and produce PGE2 (32,33). Consistently, treatment of Tregs with MC38 culture supernatant significantly induced Nr4a expression in vitro; however, this was attenuated by pre-treating MC38 cells with a COX-2 inhibitor (Fig. 4E). These data indicate that tumor-derived PGE2 actually induces Nr4a expression. In an in vivo model, Tregs from the spleen of 3LL

tumor-bearing mice showed higher Nr4a expression than that of tumor-free mice, which was abolished by administration of SC-236, a structural analogue of the clinically used COX-2 inhibitor celecoxib (Fig. 4F). Expression of the Nr4a target genes Foxp3 and

Ikzf4 was also up-regulated in Tregs in tumor-bearing mice, but was reduced by SC-236 treatment (Fig. 4F). Additionally, SC-236 treatment down-regulated Nr4a1 expression in tumor-infiltrating Tregs, and also suppressed CREB (cAMP response element binding protein) activation evaluated by the phosphorylation levels (Fig. 4G). Taken together, these results suggested that tumor-derived PGE2 induces cAMP/PKA activation, which turns on the transcription of Nr4a factors and their target genes in Tregs, and this regulatory axis can be targeted by a COX-2 inhibitor in vivo.

Camptothecin and the COX-2 inhibitor synergistically elicit anti-tumor immune responses To examine the effect of the above-identified Nr4a inhibitors on anti-tumor immunity in vivo, we treated 3LL tumor-bearing mice with CPT-11 and SC-236, according to the experimental outline shown in Fig. 5A. We found that each drug alone significantly suppressed 3LL tumor growth, and they showed more potent anti-tumor effect when used in combination (Fig. 5B). Depletion of CD8+ T cells cancelled the inhibition of

tumor growth by the inhibitors (Fig. 5C), indicating that the anti-tumor effects induced by CPT-11 and SC-236 were dependent on CD8 + T cells. A significant increase in the frequency of Ki-67+CD8+ T cells in TDLNs was observed when the mice were treated with these compounds (Fig. 5D, upper panels and Fig. 5E). Combination treatment synergistically reduced not only the Treg population but also the Foxp3 protein levels in Tregs within TDLNs (Fig. 5D, lower panels). Of note, combination therapy also synergistically enhanced production of IFN-γ from CD8+ effector T cells within the tumor (Fig. 5F, upper panels). Increase in the frequency of IFN-γ+Th1 CD4+T cells was also confirmed in the inhibitors-treated mice (Fig. 5F, lower panels). Similar results

were obtained in experiments using MC38 tumor cells (Supplementary Fig. S6A,B,C). These data support our hypothesis that pharmacological inhibition of Nr4a potently enhances anti-tumor immune responses.

Therapeutic effects of camptothecin and the COX-2 inhibitor depend on inhibition of Nr4a-mediated Treg function To gain mechanistic insights, we characterized gene expression of Tregs from TDLNs of 3LL tumor-bearing mice treated with or without Nr4a inhibitors. Expression of Nr4a1/Nr4a2 and Treg-signature genes, including Foxp3, Il2ra (Cd25), Ikzf4 (Eos), and Ctla4, were significantly down-regulated by the treatment (Fig. 6A). The suppression activity of Tregs from tumor bearing mice was higher than that of Tregs from tumor-free mice, but completely attenuated by treatment with the inhibitors (Fig. 6B and Supplementary Fig. S7A). These results indicate that Nr4a inhibitors could reverse Treg activity which was enhanced by the tumor-bearing conditions. Then to confirm that anti-tumor effects of the CPT-11/SC-236 combination depend on the inhibition of Nr4a in Tregs, we performed adoptive Treg cell transfer experiments. The 3LL tumor-bearing mice were treated with these Nr4a inhibitors, and in vitro-activated Tregs were then adoptively transferred. As shown in Fig. 6C and 6D,

WT Treg transfer significantly diminished anti-tumor effects evoked by the drugs. Conversely, transfer of Nr4a-DcKO Tregs did not reverse the therapeutic effects in the same situation (Fig. 6C,D). In addition, as observed in Nr4a-DcKO mice, the CPT-11/SC-236 treatment dramatically increased the expression levels of the co-stimulatory molecule CD80 in tumor-infiltrating DCs, which was also abolished by the transfer of WT Tregs, but not by Nr4a-DcKO Tregs (Supplementary Fig. S7B). Taken together, these data strongly support our conclusion that that the combination of CPT and a COX-2 inhibitor exerts potent anti-tumor immune responses in an Nr4a/Treg-dependent manner in vivo.

Discussion In this study, we revealed that Nr4a receptors are involved in Treg-mediated immune tolerance against tumor cells, and genetic inactivation or pharmacological inhibition of these factors could evoke CD8+ CTLs-dominant potent anti-tumor immune responses. We propose two mechanisms of CTL augmentation by Nr4a inhibition in Tregs. First is the impairment of Treg-mediated suppression of CD8+ T cell proliferation. Tregs have been shown to suppress proliferation of CD8+CTLs by depriving IL-2 since Tregs constitutively express CD25, which consists of the high-affinity Interleukin-2 receptor (34). Consistently, we observed decreased expression of CD25 in Nr4a-DcKO Tregs (Supplementary Fig. S2C,D), and reduced suppression activity compared with WT Tregs (Supplementary Fig. S2E). Therefore, reduced expression of CD25 in Nr4a-DcKO Tregs could be a mechanism for enhanced CD8+CTL expansion at the TDLNs of DcKO mice. The second mechanism is the failure of down-regulation of co-stimulatory molecules on DCs through CTLA-4. In a mouse pancreatic cancer model, Jang et al. showed that Treg depletion evokes a CD8+ CTL-dependent anti-tumor immune response in a CD11c+ DCs-dependent manner (25). We also confirmed reduced expression of CTLA-4 in Nr4a-DcKO Tregs and a drastic up-regulation of CD80 expression on tumor-infiltrating DCs in Nr4a-DcKO mice (Supplementary Fig. S3E,F,G) These observations suggest that Nr4a inhibition abolishes Treg-mediated functional control of DCs. Taken together, Nr4a factors seem to affect various aspects of tumor immunity regulated by Tregs.

In this study, we identified the classical chemotherapeutic agent CPT as an inhibitor of Nr4a transcriptional activity. It is notable that CPT treatment replicated the phenotype of Nr4a deletion in Tregs both in vitro and in vivo. However, it remains unclear how CPT inhibits Nr4a activity, for example, whether or not CPT physically

interacts with Nr4a. Although we could not find structure-activity relationships (SARs) among the drugs we screened here, amodiaquine (AQ) and chloroquine (CQ), Nr4a agonists that directly bind to the ligand-binding domain of Nr4a2 (35), contain an

quinoline skeleton similar to CPT. According to that report, AQ and CQ share a 4-amino-7-chloroquinoline entity, and other tested quinoline compounds without that structure do not affect Nr4a activities. Another report suggested that Nr4a agonists and antagonists may bind to the same site, yet the difference in the form of protein-drug interaction determines whether they work as an activator or as an inhibitor (36). Therefore, we speculate that the quinoline skeleton is important for the binding between the drug and Nr4a protein, but the structure of other skeletons or side chains in the drug may determine its function. Future studies are needed to explain the detailed mechanism of interaction between Nr4a and CPT. The COX-2/PGE2 pathway is involved in multiple aspects of tumor pathology; they directly promote tumor growth, regulate angiogenesis, and affect tumor-associated immune cells such as macrophages, myeloid-derived suppressor cells (MDSCs), and T cells (29). In this study, we demonstrated that tumor-derived PGE2 induced Nr4a factors and their target genes on Tregs in a cAMP/PKA-dependent manner, and their expression could be modulated by a common COX-2 inhibitor. Previous studies have

shown that PGE2 and its downstream cAMP/PKA pathway play important roles in the maintenance of CD4+ T cell biology, including Treg suppression activities (37-40). This is also supported by our data demonstrating that suppressive activities of Treg cells were enhanced by exposure to tumor-bearing conditions, but were attenuated by COX-2 inhibitor (Fig. 6B and Supplementary Fig. S7A). Additionally, Nr4a is well identified as a target molecule of cAMP/CREB in the signaling pathways involved in various physiological and pathological phenomena, such as hepatic glucose metabolism or the stress response in neurons (41,42). Collectively, we speculate that Nr4a factors function as a downstream regulator of PGE2-cAMP-PKA-signaling, thereby contributing to the

formation of an immunosuppressive tumor microenvironment by Tregs, and this regulatory axis can be targeted by COX-2 inhibitors. The recent success of cancer immunotherapy including immune checkpoint

blockade, such as anti-PD-1 and anti-CTLA-4 antibodies therapies, triggered the investigation of drugs that activate anti-tumor immunity from already existing drugs (43,44). Both CPT and COX-2 inhibitors were well defined as effective anti-tumor drugs directly acting on tumor cells (27,45), but here we revealed that they also enhance anti-tumor immunity by inhibiting Nr4a/Treg function. A similar finding was recently reported for cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors (13). CDK4/6 inhibitors such as abemaciclib have been shown to be effective against several solid tumors, and their primary mechanism of action is thought to be inhibition of cell cycle progression in tumor cells. Goel et al. (13) revealed that abemaciclib also promotes anti-tumor immunity by selectively suppressing the proliferation of Tregs and increasing the immunogenicity of tumor cells. Their study and our present study will accelerate the search for anti-cancer drugs that promote both tumor cell death and anti-tumor immunity. Nr4a factors may play important roles in not only Tregs but also CTLs. Mognol et al. reported that Nr4a factors are involved in the establishment of cancer-induced exhaustion in tumor-infiltrating CD8+ CTLs (46). This study suggested a possibility that Nr4a

inhibition may also facilitate the re-activation of exhausted CTLs like anti-PD-1 antibody, in addition to the inhibitory effects on Tregs. Another paper showed that Nr4a1 is involved in the function and differentiation of a long-lived subset of tissue-resident

+ + memory CD8 (TRM) T cells (47). Tumor-infiltrating CD8 T cells were reported to

exhibit characteristics of TRM cells (48). Thus, further study is necessary to elucidate the

detailed roles of Nr4a factors in anti-tumor CTLs. In conclusion, we revealed that Nr4a factors play crucial roles in various parts of Treg function in the tumor microenvironment, and these mechanisms could be applied to

tumor therapy using clinically established compounds. Nr4a factors show great promise as effective therapeutic targets of cancer immunotherapy.

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Figure Legends Figure 1. Nr4a-DcKO mice showed resistance to tumor growth (A) WT and Nr4a-DcKO mice (n= 8-12/group), and Nr4a1- and Nr4a2-single cKO

mice (n= 5/group) were subcutaneously injected with 2.5×105 3LL cells on day 0, and tumor growth was monitored. (B) Tumor growth in individual mice in (A). (C) WT and Nr4a-DcKO mice (n= 8-12/group) were subcutaneously injected with 5.0×105 MC38 cells on day 0, and tumor growth was monitored. (D) Tumor growth in individual mice in (C). (E) Representative images of MC38 tumors collected from WT and Nr4a-DcKO mice described in (C)(D) on day 22. *P < 0.05, **P < 0.01 and ***P< 0.001 compared with WT; data are presented as the mean+SD.

Figure 2. Inactivation of Nr4a receptors in Foxp3+ Tregs enhances anti-tumor immune responses (A) In vitro activated WT Tregs were transferred into 3LL tumor-bearing Nr4a-DcKO mice on days 6 and 11. Tumor weights were measured on day 18 (n= 5-7/group). (B) α-CD8 antibodies were intraperitoneally injected into 3LL tumor-bearing Nr4a-DcKO mice on days 6, 9, 13, and 16. Tumor weights were measured on day 18 (n= 5-7/group). (C-I) TDLNs (C-F) and tumor tissues (G-I) of 3LL tumor-bearing WT and Nr4a-DcKO

mice (n= 5-8 /group) were analyzed on day 20. (C) Frequency of CD4+ /CD8+ T cells. The panels show gated CD3+ T cells. (D) CD8+ T cell fraction in (C) was further analyzed for Ki-67 expression. (E) Frequency of CD4+Foxp3+ T cells. The panels show gated CD4+ T cells. (F) CD4+Foxp3+ Tregs fraction in (E) was further analyzed for Foxp3 protein expression. Numbers in the histograms indicate mean fluorescence intensities (MFIs). (G) Frequency of tumor-infiltrating CD4+/CD8+ (top) and CD4+ Foxp3+ Tregs (bottom). The lower panels show gated CD4+ T cells. (H) Ratio of tumor-infiltrating CD8+ T cells to Tregs. (I) Frequency of tumor-infiltrating effector T cells (CD8+-IFN-γ+, -TNF-α+, -CD107a+, and CD4+IFN-γ+). The panels show either

gated CD8+ or CD4+ T cells. (C-G, I) Representative FACS plots or histograms, and bar graphs summarizing the FACS data are shown. *P < 0.05 and **P < 0.01 compared with WT; n.s., not significant; data are presented as the mean+SD.

Figure 3. Identification of classical chemotherapeutic agent camptothecin as Nr4a inhibitor (A) Effects of camptothecin (CPT) on NBRE-luciferase activity transactivated by Nr4a1/Nr4a2. (B) Effects of CPT on Foxp3 promoter-luciferase activity transactivated by Nr4a2. (C) Effect of CPT on Foxp3 induction in WT naïve CD4+ T cells by ectopic expression of Nr4a2. Naïve CD4+ T cells were transduced with retroviral vectors encoding for IRES-GFP (empty) and Nr4a2-IRES-GFP (Nr4a2). CPT was added 24h after virus transduction. The panels show gated CD4+GFP+ cells. (D) Effects of CPT on Foxp3 (top) and IFN-γ expression (bottom) under iTreg and Th1 conditions, respectively. (E) Quantitative RT-PCR analysis of mRNA for the indicated genes in CD4+CD25+ WT Tregs, cultured with or without CPT in the presence of Z-VAD-fmk (50 μM) and thymidine (2 mM) for 72 h. Gene expression was normalized to 18S rRNA levels. (F) Flow cytometry profiles of the indicated proteins in WT Tregs described in (E). Dose-dependent effect of CPT was tested. (G) Effect of CPT on Foxp3 protein

expression in WT and Nr4a-DcKO Tregs treated as described in (E). One representative result (left) and data pooled from three independent experiments (right) were shown. (H) Effects of CPT on Foxp3 expression in Tregs in vivo. The CPT-derivative CPT-11 (75 mg/kg) was intraperitoneally administrated to WT mice twice at intervals of 3 days. Thymus and spleens of these mice were analyzed 6 days after the second administration (n= 5/group). (A-G) The indicated concentration of CPT was added to the culture throughout the experiment. Under culture conditions without CPT, an equivalent volume of DMSO was added only as a control. Data represent four (A-F) or three (G) independent experiments. As for (A,B, E), each experiment was performed in triplicate.

(C,D, F, H) Representative FACS plots or histograms, and bar graphs summarizing the FACS data are shown. Numbers in the histograms of (F) and (H) indicate mean fluorescence intensities (MFIs). For the bar chart in (F) and (G), MFIs of CPT-untreated

WT Tregs are set as one. *P < 0.05, **P < 0.01, ***P< 0.001 compared with the untreated group unless otherwise indicated; n.s., not significant; data are presented as the mean+SD.

Figure 4. COX-2 inhibitor targets PGE2-Nr4a axis on Tregs in tumor microenvironment (A-E) RNA was purified from the CD4+CD25+ Tregs cultured under the in vitro conditions described below, and mRNA expression of the indicated genes was measured by quantitative RT-PCR. (A) WT Tregs were serum-starved for 1 h, then stimulated with the indicated concentration of PGE2 for 3 h. Dose-dependent effect of PGE2 was tested. (B) Tregs were serum-starved for 1 h, then stimulated with PGE2 (25 μM) for 3 h following pretreatment with DMSO vehicle or PKA inhibitor H-89 (10 μM) for 1 h. (C) Tregs were cultured with or without 25 μM PGE2 and H-89 (10 μM) under TCR stimulation for 18 h. (D) WT or Nr4a-DcKO Tregs were cultured as described in

(C). Expression levels of PGE2-untreated Tregs are set as one. (E) WT Tregs were incubated with control media or tumor cell supernatant (TSN) under TCR stimulation for 18 h. TSN was derived from MC38 tumor cells cultured in the presence or absence of the COX-2 inhibitor SC-236 (10 μM). (F) Quantitative RT-PCR analysis of mRNA for the indicated genes in CD4+CD25+ WT Tregs, isolated from spleens of tumor-free (no tumor) and 3LL tumor-bearing mice treated with or without SC-236. Tregs from three mice per group were pooled for one sample. Intraperitoneal administration of SC-236 (3 mg/kg) was performed on days 6 and 9, and mice were analyzed on day 14. (G) Nr4a1 expression and phosphorylation level of CREB on tumor-infiltrating Tregs

from 3LL tumor-bearing mice treated with or without SC-236 (n= 4/group) as described in (F). Representative histograms and bar graphs summarizing the FACS data are shown. Numbers in the histograms indicate mean fluorescence intensities (MFIs). FMO;

Fluorescence minus one. (A-E) Under culture condition without PGE2, an equivalent volume of ethanol was added only as a control. (A-F) Gene expression was normalized to 18S rRNA levels. Data were representative of three independent experiments each performed in triplicate. *P < 0.05, **P < 0.01, ***P< 0.001 compared with the untreated group unless otherwise indicated ; n.s., not significant; data are presented as the mean+SD.

Figure 5. Combination therapy with camptothecin and COX-2 inhibitor SC-236 synergistically induced potent anti-tumor immune responses (A) Schematic of the experimental design. WT mice were subcutaneously injected with 2.5×105 3LL tumor cells (day 0). CPT-11 (75 mg/kg) and/or SC-236 (3 mg/kg) were intraperitoneally injected on days 6 and 9. (B-F) 3LL tumor-bearing mice were left untreated or treated as described in (A), and analyzed on day 14 (n= 6-8/group). (B) Tumor growth was monitored throughout the experimental period. (C) α-CD8

antibodies were intraperitoneally injected on days 5, 7, and 10. Tumor weights were measured. (D) Frequency of CD4+/CD8+ (top) and CD4+Foxp3+ (bottom) T cells in TDLNs. The panels show either gated CD3+ or CD4+ cells, respectively. The rightmost histogram shows gated CD4+Foxp3+ cells, and the numbers indicate mean fluorescence intensities (MFIs) of Foxp3. (E) The CD3+CD8+ T cell fraction in (D) was further analyzed for Ki-67 expression. (F) Frequency of tumor-infiltrating effector T cells (CD8+IFN-γ+ and CD4+IFN-γ+). The panels show either gated CD8+ or CD4+ cells. (D-F) Representative FACS plots and bar graphs summarizing the FACS data are shown. *P < 0.05; **P < 0.01, ***P < 0.001 compared with the untreated group unless

otherwise indicated; data are presented as the mean+SD.

Figure 6. Therapeutic effects of camptothecin and the COX-2 inhibitor SC-236 are

dependent on inhibition of Treg function governed by Nr4a (A,B) 3LL tumor-bearing WT mice were left untreated or treated with combination of CPT-11/SC-236 as described in Fig. 5A, followed by isolation of TDLN Tregs. Data are representative of three independent experiments involving four to six mice per group. (A) Quantitative RT-PCR analysis of mRNA for the indicated genes. Gene expression was normalized to 18S rRNA levels. Each experiment was performed in triplicate. (B) In vitro suppression assay. Suppression of CFSE-labeled CD4+CD25- CD45.1+ cells (responder T cells; Tresp) by CD45.1- Tregs from tumor-free (no tumor), and 3LL tumor-bearing mice treated with or without inhibitors. Cells were stimulated with anti-CD3/28 beads for 96 h. Numbers in histograms represent percentages of undivided cells gated on CD4+CD45.1+. (C,D) 3LL tumor-bearing mice were left untreated or treated with combination of CPT-11/SC-236 as described in Fig. 5A. On the day after drug administration (day 10), in vitro activated WT or Nr4a-DcKO Tregs were transferred. These mice were analyzed on day 15 (n= 5-8/group). (C) Tumor weights. (D) Frequency of tumor-infiltrating effector T cells (CD8+-IFN-γ+, -TNF-α+, -CD107a+).

The panels show gated CD8+ T cells. Representative FACS plots and bar graphs summarizing the FACS data are shown. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the untreated group; n.s., not significant; data are presented as the mean+SD

Inhibition of Nr4a receptors enhances anti-tumor immunity by breaking Treg-mediated immune tolerance

Sana Hibino, Shunsuke Chikuma, Taisuke Kondo, et al.

Cancer Res Published OnlineFirst March 20, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-17-3102

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