Published OnlineFirst October 13, 2016; DOI: 10.1158/0008-5472.CAN-16-1140 Cancer Therapeutics, Targets, and Chemical Biology Research Biphasic Rapamycin Effects in Lymphoma and Carcinoma Treatment Yang Liu1,2,3,4, Srilakshmi Pandeswara2, Vinh Dao1,2, Alvaro Padron 2, Justin M. Drerup1, Shunhua Lao2, Aijie Liu2, Vincent Hurez2, and Tyler J. Curiel1,2,3 Abstract mTOR drives tumor growth but also supports T-cell function, memory T cells in EL4 challenge, but without clinical benefit. LD rendering the applications of mTOR inhibitors complex especially rapamycin significantly enhanced DD treatment efficacy, but DD in T-cell malignancies. Here, we studied the effects of the mTOR plus LD rapamycin treatment effects were independent of anti- inhibitor rapamycin in mouse EL4 T-cell lymphoma. Typical tumor immunity. Instead, rapamycin upregulated EL4 IL2 recep- pharmacologic rapamycin (1–8 mg/kg) significantly reduced tor in vitro and in vivo, facilitating direct DD tumor cell killing. tumor burden via direct suppression of tumor cell proliferation LD rapamycin augmented DD efficacy against B16 melanoma and improved survival in EL4 challenge independent of antitu- and a human B-cell lymphoma, but not against human Jurkat mor immunity. Denileukin diftitox (DD)–mediated depletion T-cell lymphoma or ID8agg ovarian cancer cells. Treatment of regulatory T cells significantly slowed EL4 growth in vivo in a effects correlated with IL2R expression, but mechanisms in T-cell–dependent fashion. However, typical rapamycin inhibited some tumors were not fully defined. Overall, our data define T-cell activation and tumor infiltration in vivo and failed to boost a distinct, biphasic mechanisms of action of mTOR inhibition DD treatment effects. Low-dose (LD) rapamycin (75 mg/kg) at doses that are clinically exploitable, including in T-cell þ þ increased potentially beneficial CD44hiCD62L CD8 central lymphomas. Cancer Res; 77(2); 1–12. Ó2016 AACR. Introduction type of B-cell lymphoma, but there are few studies of their clinical effects in T-cell lymphomas. The serine/threonine kinase, mTOR, is an important regulator Aside from inhibiting tumor mTOR to slow tumor growth of cell growth, metabolism, and stress responses (1) with directly, mTOR inhibition also augments conventional (11, 12) increased activation in many cancers (2), including B-cell and and gd T-cell (13) functions that can affect anticancer immunity T-cell hematologic malignancies (3, 4). Treating cancers with (14). We show here that rapamycin has bimodal and novel rapamycin (sirolimus), a small-molecule mTOR inhibitor, or treatment effects against murine EL4 T-cell lymphoma. At typical related mTOR inhibitors collectively called rapalogues, is thus pharmacologic doses, rapamycin inhibited tumor mTOR and a reasonable cancer treatment strategy. Rapalogues are approved slowed EL4 lymphoma growth in vivo through direct effects on to treat certain carcinomas, but with generally modest efficacy (5). tumor cells, but reduced immune T-cell activation. Low-dose (LD) Human B-cell (6) and T-cell (4, 7) malignancies express acti- rapamycin improved antitumor immunity against EL4 when vated mTOR that can drive their growth. Distinct rapalogues have combined with a vaccine plus immune checkpoint blockade shown efficacy against them in vitro or in immunocompromised (14). However, we found that LD rapamycin did not affect in mice (8, 9), suggesting direct antitumor effects. Rapalogues vivo EL4 growth, minimally inhibited tumor mTOR, but did not have some clinical activity in mantle cell lymphoma (9, 10), a blunt immune T-cell activation in vivo. EL4 was refractory to many immunotherapies but responsive to regulatory T-cell (Treg) depletion. We tested the IL2/diphtheria 1The Graduate School of Biomedical Sciences, University of Texas Health Science toxin fusion protein, denileukin diftitox (DD; refs. 15, 16), as Treg Center, San Antonio, Texas. 2Department of Medicine, University of Texas Health depletion immunotherapy and found that typical rapamycin Science Center, San Antonio, Texas. 3Cancer Therapy and Research Center, doses did not improve Treg depletion as immunotherapy, as 4 University of Texas Health Science Center, San Antonio, Texas. Xiangya School T-cell activation was blunted. By contrast, LD rapamycin of Medicine, Central South University, Changsha, Hunan, P.R. China. improved DD treatment efficacy, but unexpectedly, this LD effect Note: Supplementary data for this article are available at Cancer Research did not require antitumor immunity. Instead, rapamycin Online (http://cancerres.aacrjournals.org/). increased tumor expression of IL2 receptor (IL2R) that improved Current address for Y. Liu: Department of Emergency, Peking Union Medical killing of tumor cells by DD, which binds this receptor. We used College Hospital, Chinese Academy of Medical Sciences, Beijing, 100730, China. mouse B16 melanoma, ID8agg ovarian carcinoma, human Corresponding Author: Tyler J. Curiel, University of Texas Health Science NM001 EBV-transformed B-cell lymphoma and human Jurkat þ Center, Adult Cancer Program STRF MC8252, 8403 Floyd Curl Drive, San CD4 T-cell lymphoma lines in additional rapamycin studies, Antonio, TX 78229-3904. Phone: 210-450-1438; Fax: 210-450-1234; E-mail: where we found that rapamycin improved DD efficacy only in [email protected] IL2R–expressing tumor cells. The mTOR inhibitor, temsirolimus doi: 10.1158/0008-5472.CAN-16-1140 improved DD-mediated cytotoxicity against IL2R–expressing Ó2016 American Association for Cancer Research. human B-cell lymphoma line NM001, but not against the Jurkat www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst October 13, 2016; DOI: 10.1158/0008-5472.CAN-16-1140 Liu et al. þ human CD4 T-cell line lacking IL2R. We thus demonstrate an RMP1-14), or aCTLA-4 (clone 9H10, all from BioXCell) mono- unexpected mechanism for mTOR inhibitor use in cancer com- clonal antibodies intraperitoneally on days 4, 7 and 12. aCD25 bination immunotherapy and show distinct, biphasic mechan- (250 mg, clone PC-61.5.3, BioXCell) was administered intra- isms of rapamycin action at typical versus LDs of rapamycin, with peritoneally on day 2. Denileukin diftitox (DD, Eisai), a fusion similar temsirolimus effects. protein of diphtheria toxin and human IL2 (18), that depletes mouse Tregs to treat distinct carcinomas (15, 16, 19), was given Materials and Methods 5 mg i.p. every 4 days for EL4 challenge, and every 5 days for B16 and ID8agg challenge. Foxp3DTR mice engineered to allow Mice Treg-specific depletion using diphtheria toxin (15) were Wild type (WT), male bd TCR (T-cell receptor) knockout (KO), injected intraperitoneally with 1 mg/kg diphtheria toxin every interferon-g KO, d TCR KO, Rag2 (recombinase activating gene 2) 3 days starting on day 4. Rapamycin (LC Laboratories) or KO, Prf1 (perforin) KO on the C57BL/6J background (BL6) were vehicle control (0.25% Tween 80 þ 0.25% PEG400) was given purchased from The Jackson Laboratory. Male BL6 Foxp3DTR mice intraperitoneally daily for 5 consecutive days per week at were from Alexander Rudensky, University of Washington and indicated doses starting on day 4, for times indicated. Rapa- used for studies shown. Female WT, bd TCR KO and Foxp3DTR mice mycinat1to8mg/kgisdefined as typical, and 0.075 mg/kg as were used as noted. Mice were maintained in specific pathogen-free LD. conditions, provided a normal diet and water ad libitum and used when 8- to 16—weeks-old. All animal studies were approved by Flow cytometry our Institutional Animal Care and Use Committee. Tumors were isolated and digested with 1.67 Wunsch€ U/mL Liberase TL (Roche) and 0.2 mg/mL DNase I (Roche) in RPMI- Cell lines 1640 at 37C for 30 minutes. Tumors, tumor-draining lymph Mouse EL4 lymphoma, B16F10 melanoma and human Jurkat þ nodes (TDLN), or spleens were stained and analyzed on a BD LSR CD4 T-cell lymphoma cells were purchased from the ATCC in II flow cytometer using FACSDiva software. For intracellular 2012, 2008, and 1987, respectively. Mouse ID8 was a gift from staining, cells were fixed and permeabilized with Foxp3/transcrip- George Coukos (University of Pennsylvania) in 2004, from which tion factor buffer (eBioscience) per manufacturer's directions. we developed the highly aggressive ID8agg subline (our unpub- Anti-mouse antibodies were against CD4 (Tonbo); CD8a (Life lished data). NM001 is a human EBV-transformed B cell lym- Technologies); B7-H1, CD132 (BD Biosciences); Foxp3, Ki-67, phoma line generated as we described (17) in 2015. Cell lines Vb12 TCR, ICOS (eBioscience); and CD3, CD25, CD44, CD62L, were not independently validated. All murine lines are on the BL6 CD122, PD-1, Annexin V (Biolegend). Antibodies against human background and all cells were cultured in medium RPMI-1640 CD25, CD122 and CD132 were from Biolegend. We used Ghost containing 4 mmol/L L-glutamine, 100 U/mL penicillin and 100 Dye UV 450 viability dye (Tonbo), and Dead Cell Apoptosis Kit mg/mL streptomycin, 10 mmol/L HEPES, 1 mmol/L sodium with Annexin V Alexa Fluor 488 and propidium iodide (Invitro- þ þ þ pyruvate and 10% FBS, in a 5% CO2, humidified atmosphere at gen). In the CD3 gate, EL4 cells were GFP Vb12 and distin- À À 37 C. The pAcGFP1-C1 plasmid was used to generate EL4 cells guished from resident GFP Vb12 T cells. In specific experiments, stably expressing GFP (EL4-GFP) using the Cell Line Nucleofector EL4 cells were stimulated in 96-well round-bottom plates using Kit (Lonza) according to the manufacturer's instructions. Forty- CD3/CD28 beads (Dynabeads Mouse T-cell activator, Invitrogen) eight hours after transfection, cells expressing GFP were sorted for 24 hours at 1 bead:5 EL4 cells, before staining for CD25 and fl using a BD FACSAria ow cytometer with FACSDiva software (BD CD122. Bioscience), and then maintained in medium containing G418 (1 mg/mL) for selection. ID8agg cells expressing luciferase were Immunoblotting generated with the luciferase-encoding plasmid pGL4.51 (Pro- Snap-frozen tumor tissues were homogenized in RIPA buffer mega) with Attractene transfection reagent and selected with (20 mmol/L Tris–HCl pH 8.0, 150 mmol/L NaCl, 1 mmol/L G418 (1 mg/mL).