Stat3 Inhibition Augments the Immunogenicity of B-Cell Lymphoma Cells, Leading to Effective Antitumor Immunity

Published OnlineFirst June 22, 2012; DOI: 10.1158/0008-5472.CAN-11-3619

Cancer Therapeutics, Targets, and Chemical Biology Research

Stat3 Inhibition Augments the Immunogenicity of B-cell Lymphoma Cells, Leading to Effective Antitumor Immunity

Fengdong Cheng1, Hongwei Wang1, Pedro Horna1,3, Zi Wang1, Bijal Shah1, Eva Sahakian1, Karrune V. Woan1, Alejandro Villagra1, Javier Pinilla-Ibarz1, Said Sebti2, Mitchell Smith4, Jianguo Tao1,3, and Eduardo M. Sotomayor1

Abstract Mantle cell lymphoma (MCL) is an aggressive and incurable subtype of B-cell non–Hodgkin lymphomas. Although patients often respond initially to first-line treatment with chemotherapy plus monoclonal antibodies, relapse and decreased response to further lines of treatment eventually occurs. Harnessing the immune system to elicit its exquisite specificity and long-lasting protection might provide sustained MCL immunity that could potentially eradicate residual malignant cells responsible for disease relapse. Here, we show that genetic or pharmacologic disruption of Stat3 in malignant B cells augments their immunogenicity leading to better þ activation of antigen-specific CD4 T cells and restoration of responsiveness of tolerized T cells. In addition, treatment of MCL-bearing mice with a specific Stat3 inhibitor resulted in decreased Stat3 phosphorylation in malignant B cells and anti-lymphoma immunity in vivo. Our findings therefore indicate that Stat3 inhibition may represent a therapeutic strategy to overcome tolerance to tumor antigens and elicit a strong immunity against MCL and other B-cell malignancies. Cancer Res; 72(17); 1–9. 2012 AACR.

Introduction cell non–Hodgkin lymphomas (NHL; ref. 6). As such, novel – Previous studies in murine models of B-cell lymphoma non cross-resistant treatment modalities capable of improv- indicate that generation of effective anti-lymphoma immunity ing the response rate and more importantly able of sustaining requires (i) conversion of bone marrow–derived antigen-pre- these responses are greatly needed for patients with MCL. senting cells (APC) from a noninflammatory (or tolerogenic) Several lines of evidence point to manipulation of the – status into inflammatory APCs that trigger effective T-cell immune system as an enticing non cross-resistant therapeutic responses (1, 2) and (ii) augmentation of the APC function of strategy for MCL. The demonstration that immune cells are the malignant B cells (3). Therapeutic strategies endowed with able to kill chemotherapy-resistant tumor cells (7, 8) together fi the ability of fulfilling both requirements might not only lead to with the ndings that T-cell responses can be elicited in successful eradication of B-cell tumors but also to a long- vaccinated patients with MCL (9), and the encouraging lasting immunity, and the latter is a desirable effect for certain responses observed in patients with relapsed/refractory MCL B-cell malignancies characterized by their high tendency to treated with immunomodulatory drugs (10, 11) suggest that relapse. harnessing the immune system and, in particular, eliciting its fi Mantle cell lymphoma (MCL) is the prototype of a B-cell exquisite speci city and long-lasting protection might lead to malignancy in which relapse is the major challenge to over- sustained immune responses in MCL (12). fi come. In spite of a good initial response to first-line treatment Given the above rationale, a signi cant effort has been fl with chemotherapy plus monoclonal antibodies, almost all devoted to identify molecular target(s) capable of in uencing fl patients with MCL will eventually relapse, becoming less in ammatory pathways in APCs as well as in malignant B cells. responsive to further lines of treatment and ultimately will Stats are cytoplasmic transcription factors that are key med- succumb to their disease (4, 5). Given these sobering char- iators of cytokine and growth factor signaling pathways (13). acteristics, MCL has one of the worst prognoses among all B- One of the members of the Stat family, Stat3, has emerged as a negative regulator of inflammatory responses in a variety of immune cells (14–16). For instance, we have previously shown Authors' Affiliations: Departments of 1Malignant Hematology, 2Drug that pharmacologic or genetic disruption of Stat3 in APCs Discovery, and 3Hematopathology, H. Lee Moffitt Cancer Center & resulted in diminished production of the anti-inflammatory Research Institute, Tampa, Florida; and 4Fox Chase Cancer Center, Phil- adelphia, Pennsylvania cytokine IL-10, enhanced expression of costimulatory molecules, and increased release of proinflammatory mediators Corresponding Author: Eduardo M. Sotomayor, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Drive, FOB-3, Room 5.3125, leading to augmentation of the function of these cells to Tampa, FL 33612. Phone: 813-745-1387; Fax: 813-745-3071; E-mail: effectively prime T cells and restore the responsiveness of þ Eduardo.Sotomayor@moffitt.org anergic CD4 T cells (17). These observations prompted us doi: 10.1158/0008-5472.CAN-11-3619 to ask whether targeting Stat3 in malignant B cells might also 2012 American Association for Cancer Research. influence the immunogenicity and inflammatory status of

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these cells and whether such an effect might unleash effective Stat3c expression vector or control vector. Inhibition of Stat3 in antitumor immune responses in a murine model of MCL. JEKO human MCL was accomplished with siRNA specific for Stat3 using Amaxa Nucleofector methodology per manufac- Materials and Methods turer's protocol (Dharmacon). Mice Six-week-old male BALB/c (H-2d) and C57BL/6 (H-2b) mice Isolation of malignant B cells in vivo were obtained from the NIH (Frederick, MD). Male BALB/c Mice were sacrificed and tumor nodules were carefully severe combined immunodeficient (SCID) or C57BL/6 SCID dissected from their livers. Tumors were gently mashed in mice, aged 6 weeks, were purchased from Jackson Laborato- tissue culture plates and cells were transferred to a conical tube ries. T-cell receptor (TCR) transgenic mice expressing an ab T- and washed twice in RPMI-1640. Cells were cultured for 3 hours cell receptor specific for amino acids 110 to 120 from influenza at 37 C, 5% CO2, and nonadherent cells were collected for hemagglutinin presented by I-Ed were a gift by H. von Boehmer further experiments. (18). TCR transgenic mice (OT-II) expressing an ab TCR- specific for peptide 323–339 from ovalbumin (OVA) presented Immunoblotting by MHC class II and I-Ab (19) were provided by Dr. W. Heath Whole-cell lysates were prepared using modified radio- (The Walter and Eliza Hall Institute of Medical Research, immunoprecipitation assay (RIPA) lysis buffer. Fifty micro- Victoria, Australia). All experiments involving the use of mice grams of protein was subjected to SDS-PAGE and transferred were carried out in accordance with protocols approved by the onto polyvinylidene difluoride (Millipore) membranes and Animal Care and Use Committees of the University of South incubated overnight with primary antibodies, followed by Florida College Of Medicine (Tampa, FL). horseradish peroxidase–conjugated secondary antibodies (Pierce) and finally, proteins were visualized with a Chemi- Tumor cells luminescent Detection Kit (Pierce). Primary antibodies Murine A20 lymphoma cells (H-2d)andhumanJEKOMCL against phospho-Stat3 (Tyr705), phospho-AKT, and phos- cells were obtained from American Type Culture Collec- pho-p42/44 MAPK were purchased from Cell Signaling Tech- tion. A20 lymphoma cells expressing HA (Hemagglutinin nology. Anti-Stat3 and anti-AKT antibodies were purchased influenza) as a model tumor antigen were selected and from Santa Cruz Biotechnology and anti-glyceraldehyde- grown in vitro as previously described (20). FC-muMCL1 3-phosphate dehydrogenase was purchased from Sigma cell line (H-2b) was derived from a tumor-explanted, 1-year- (Sigma-Aldrich). old Bcl-1 transgenic mice injected with pristane intraperi- toneally (i.p.; ref. 21). Cells were cultured in RPMI-1640 In vitro and in vivo pharmacologic inhibition of Stat3 medium supplemented with 15% FBS. All cell lines were CPA-7 is a platinum-containing compound that disrupts evaluated by flow cytometry for characterization before Stat3 DNA-binding activity, but not Stat5 nor Stat1 in malig- experimentation. For in vivo experiments, cells were washed nant cells (25). For in vitro studies, FC-muMCL1 cells were 3 times in sterile Hank's Balanced Salt Solution (HBSS) and treated with CPA-7 alone (30–1,000 nmol/L) or in combination then 1 106 A20 or 5 106 FC-muMCL1 cells were inject- with LPS (2 mg/mL), and their ability to present cognate þ ed either subcutaneously or i.p. into BALB/c or C57BL/6 peptide to antigen-specific CD4 T cells was determined as mice, respectively. described under In vitro antigen presentation studies. For in vivo studies, FC-muMCL1 or A20 tumor–bearing mice were Reagents given CPA-7 i.v. at the dose of 5 mg/kg every 3 days as LPS (Escherichia coli 055:B5, L-2880) was purchased from previously described (26). Sigma-Aldrich. CPA-7 was provided by S. Sebti (H. Lee Moffitt Cancer Center, Tampa, FL). CPA-7 was first reconstituted in In vivo generation of anergized CD4 T cells þ dimethyl sulfoxide (DMSO) and then further diluted in culture Briefly, 2.5 106 CD4 transgenic T cells specific for an MHC medium for in vitro or in HBSS for in vivo use. class II epitope of HA were injected i.v. into A20HA lymphoma– bearing mice. Twenty-one days after T-cell transfer, animals Transfection of tumor cells were sacrificed and anergized T cells were reisolated from their A20 B cells were transfected with either a dominant-negative spleens as previously described (20). Cytokine production by þ variant of Stat3, Stat3b (22, 23), or Stat3c, a mutant form of reisolated clonotypic CD4 T cells in response to HA Stat3 that is constitutively activated without tyrosine phos- peptide110–120 presented by A20 B cells was determined as phorylation (24). Transfections were conducted via electropo- described under antigen presentation studies. ration according to the manufacturer's instructions (Bio-Rad). For induction of antigen-specific T-cell tolerance in H-2b Briefly, A20 B cells were harvested and washed with cold PBS, tumor–bearing mice, a similar experimental approach was þ then resuspended at the concentration of 1 107/0.3 mL in used, the only difference being that 1 106 anti-OVA CD4 PBS and transferred into an electroporation cuvette. Then, 15 transgenic T cells (OT-II) were transferred into animals bearing mg of either GFP, Stat3b GFP DNA, or PBS was added and cells an ovalbumin-expressing tumor (B16OVA). Fourteen days were subjected to a high-voltage electrical pulse of defined after T-cell transfer, animals were sacrificed and anergized magnitude and length as per manufacturer's instructions. A OT-II cells were reisolated from their spleens (17). Cytokine similar procedure was followed to transfect A20 cells with a production by OT-II cells in response to OVA peptide323–339

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presented by FC-muMCL1 cells was determined as described below.

In vitro antigen presentation studies A20 or FC-muMCL1 cells (1 105 per well) were cultured þ with 5 104 puriﬁed naive or tolerized antigen-speciﬁc CD4 T cells in the presence or absence of cognate peptide (either synthetic HA peptide110–120 SFERFEIFPKE for studies with A20 B cells or OVA peptide323–339, ISQAVHAAHAEINEAGR for studies with FC-muMCL1 cells; American Peptide Company) After 48 hours, supernatants were collected and assayed for IL- 2 and IFN-g production by ELISA (R&D Systems). Values for T cells cultured in media alone were routinely less than 10% of the values for antigen-stimulated T cells.

Flow cytometric analysis FC-muMCL1 cells were stained with phycoerythrin (PE) anti-CD5 (53–7.3; BD Bioscience), PE-Cy7 anti-CD19 (1D3; BD Figure 1. Disruption of Stat3 in malignant B cells augments antigen- þ Bioscience), and fluorescein isothiocyanate anti-cyclin D1 specific CD4 T-cell responses. A20 B cells were untransfected (None), mock transfected (Mock), or transiently transfected with GFP or Stat3b (DCS-6, Millipore) antibodies. The expression of B7.1, B7.2, GFP expression vector. Then, 1 105 transfected or control cells were CD40, and MHC class I and II in FC-muMCL1 and A20 B cells incubated with either 5 104 naive (left) or tolerized anti-HA CD4þ T cells was determined using specific antibodies (16-10A1, GL1, 1C10, isolated from the spleen of A20HA-bearing mice (right) in the presence of 28-14-8, M5/114.15.2; eBiosience). Fifty thousand gated events 12.5 mg of HA peptide110–120 SFERFEIFPKE. After 48 hours, supernatants were collected on a FACSCalibur (BD Biosciences) and ana- were collected and assayed for IL-2 (A and C) and IFN-g (B and D) by ELISA. Values represent mean SE of triplicate cultures and are lyzed using FlowJo software (Tree Star). representative of 3 independent experiments (, P < 0.05 statistically significant for the difference in cytokine production). Statistical analysis A 2-way ANOVA was used to evaluate the magnitudes of incubation of these same tumor-anergized T cells with A20- cytokine production by clonotypic T cells. Differences in Stat3b lymphoma cells resulted in restoration of T-cell respon- survival were assessed with the log-rank test. siveness to cognate HA-antigen. Indeed, presentation of HA- peptide by A20-Stat3b triggered IL-2 (Fig. 1C) and IFN-g þ Results production by tolerant CD4 T cells (Fig. 1D). In sharp contrast, anergic T cells encountering HA antigen on nontrans- Genetic manipulation of Stat3 signaling in malignant B fected, mock-transfected, or GFP-transfected A20 B cells fl fi in vitro cells in uences antigen-speci c T-cell responses remained unresponsive. First, we asked whether genetic manipulation of Stat3 in A20 fi fl Given the above ndings, we next asked whether an opposite lymphoma B cells could in uence their intrinsic antigen-pre- effect would be observed when Stat3 is overexpressed in senting capabilities and the responsiveness of antigen-specific þ malignant B cells. A20 cells were therefore transfected with CD4 T cells. To inhibit Stat3 in A20 cells, we used a dominant- Stat3c, a mutant form of Stat3 that is constitutively activated negative variant of Stat3, Stat3b (22). A20 cells were transfected without tyrosine phosphorylation (24). Unlike naive anti-HA in vitro þ with Stat3b (A20-Stat3b) and then cultured with CD4 T cells that produce IL-2 and IFN-g in response to þ fi syngeneic naive CD4 T cells speci c for an MHC class II cognate antigen presented by control A20 B cells (Fig. 2A and þ restricted epitope of HA in the presence or absence of cognate B, non-transfected or PC-DNA transfected), CD4 T cells HA-peptide. As shown in Fig. 1 (left), clonotypic T cells cultured with A20-Stat3c cells were rendered unresponsive encountering HA-peptide on A20-Stat3b cells displayed a fi given their minimal production of IL-2 (Fig. 2A, Stat3c) and signi cantly enhanced production of IL-2 (Fig. 1A) and IFN- inability to produce IFN-g in response to cognate antigen (Fig. g (Fig. 1B), relative to those T cells encountering cognate 2B, Stat3c). Taken together, genetic manipulation of Stat3 in peptide on either nontransfected (None), mock-transfected A20 B cells determines the functional outcome of antigen- (Mock), or GFP-transfected (GFP) A20 B cells. specific T cells and points to Stat3 inhibition as an enticing In previous studies, we have shown that adoptive transfer of þ approach to overcome T-cell anergy. naive anti-HA transgenic CD4 T cells into mice bearing A20 B cell lymphoma, expressing HA as a model tumor antigen þ (A20HA), resulted in the induction of antigen-specific CD4 Pharmacologic inhibition of Stat3 in murine B-cell T-cell tolerance. In this system, reisolated T cells from lym- lymphomas augments APC function phoma-bearing mice were found to be anergic by their failure Given that inhibition of Stat3 in malignant B cells induces þ to be primed in vivo as well as by their diminished IL-2 and IFN- better activation of antigen-specific CD4 T cells and restores g production in response to in vitro restimulation with cognate the responsiveness of tolerized T cells, we asked next whether HA-peptide (20). However, as shown in Fig. 1 (right), in vitro this positive effect was due to the augmentation of the APC

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all C57BL/6 mice challenged with 5 106 FC-muMCL1 given either subcutaneously (black circle) or i.p. (open circle) developed tumors and showed decreased survival (Fig. 4B). In mice challenged subcutaneously, tumor nodules developed by day 21. Intraperitoneal injection of MCL cells resulted in the development of ascites by day 30 and at necropsy we found enlarged mesenteric and retroperitoneal lymph nodes as well as tumor nodules in the peritoneum and small bowel (data not shown). Next, we determined the in vitro effects of CPA-7 upon FC- muMCL1 cells. First, CPA-7 did not affect the viability or proliferation of malignant B cells (Supplementary Fig. S1). Figure 2. Increased Stat3 activity in malignant B cells inhibits antigen- þ Second, we assessed whether FC-muMCL1 cells can present specificCD4 T-cell responses. A20 B cells were transiently transfected fi þ with either pcDNA3 empty vector or Stat3c expression vector. Then, cognate antigen to antigen-speci c CD4 T cells and if so, 1 105 transfected or control cells were incubated with 5 104 naive whether this intrinsic APC function can be enhanced by CPA-7. anti-HA CD4þ T cells in the presence or absence of 12.5 mgofHApeptide. Given the background of FC-muMCL1 cells (H-2b), we assessed þ After 48 hours, supernatants were collected and assayed for IL-2 (A) and their ability to present ovalbumin peptide to transgenic CD4 IFN-g (B) production by ELISA. Values represent mean SE of triplicate fi T cells expressing a abTCR speci c for OVA peptide323–339 (19). cultures and are representative of 3 independent experiments ( , P < 0.05 þ statistically significant for the difference in cytokine production). First, anti-OVA CD4 T cells encountering cognate antigen on untreated MCL cells (ovalbumin peptide) or in LPS-treated function of malignant B cells. Therefore, the expression of MHC MCL cells (LPSþOVA) produced IL-2 (Fig. 4C, left) and IFN-g and costimulatory molecules was determined in A20 B cells (Fig. 4C, right) indicative of the ability of murine MCL cells to þ treated in vitro with CPA-7, a platinum-containing Stat3-spe- present antigen to CD4 T cells in vitro. This intrinsic APC cific inhibitor (25). As shown in Fig. 3, CPA-7–treated A20 B function was enhanced following exposure of MCL cells to LPS cells displayed elevated expression of the costimulatory mole- in the presence of increasing concentrations of Stat3 inhibitor. cules B7.1, B7.2, and CD40. No significant changes in the Indeed, CPA-7–treated MCL cells triggered an increased pro- þ expression of MHC class I or II molecules were observed duction of IL-2 and IFN-g by CD4 T cells (Fig 4C; CPA-7þLPS). þ among untreated or CPA-7–treated malignant B cells (data Importantly, anergized anti-OVA CD4 T cells cultured in not shown). However, the expression of MHC class I and II were vitro with CPA-7–treated FC-muMCL1 cells regained their increased in A20 cells treated with CPA-7 plus LPS in com- ability to produce IFN-g in response to cognate ovalbumin parison with cells treated with LPS alone (data not shown). peptide (Fig. 4D, CPA-7þLPS). In contrast, anergized T cells Recently, Smith and colleagues have developed a murine encountering ovalbumin peptide on untreated MCL cells model of MCL by injecting pristane i.p. into 1-year-old Bcl-1 (OVA) or in cells treated with LPSþOVA peptide (in the transgenic mice (Em-cyclin D1).In these animals, the pattern of absence of CPA-7) remained unresponsive (Fig. 4D). Finally, disease consists of diffuse adenopathy, splenomegaly, bone reminiscent of our observations in CPA-7–treated A20 B cells, marrow infiltration as well as lung, kidney, and periportal FC-muMCL cells treated with CPA-7 also displayed enhanced hepatic infiltration. Analysis of tumor explants revealed malig- expression of the costimulatory molecules B7.1 and B7.2 nant B cells that coexpress cyclin D1, CD20, and CD5, but lack relative to untreated cells (Fig. 4E). However, no changes in expression of CD23, findings reminiscent of human MCL (21). the expression of CD40 was observed in CPA-7–treated cells A cell line, FC-muMCL1, has been derived from one of these relative to controls (data not shown). Taken together, treat- lymphoma explants and phenotypic analysis confirmed that ment of murine MCL cells with CPA-7 augments their immu- þ they express cyclin D1, CD19, and CD5 (Fig. 4A). Furthermore, nogenicity resulting in enhanced activation of naive CD4 T

Figure 3. Enhanced expression of costimulatory molecules on malignant B cells treated with CPA- 7. Expression of B7.1, B7.2, and CD40 on A20 B cells treated or not with CPA-7 (15 mmol) for 15 hours was assessed by ﬂow cytometry. Fifty thousand gated events were collected on a FACSCalibur and analyzed using FlowJo software. Gray histogram shows isotype control. Shown are representative of 3 experiments with similar results.

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Figure 4. CPA-7 augments the antigen presenting function of FC-muMCL1 cells. A, expression of cyclin D1, CD19, and CD5 by FC-muMCL1 cells (open histogram) was assessed by flow cytometry. Gray histogram shows isotype control. B, in vivo growth of FC-muMCL1 tumors was determined in C57BL/6 mice injected either subcutaneously or i.p. with 5 106 FC-muMCL1 cells. Five mice were included in each group and inspected 3 times a week for the development of tumor. Shown is a representative experiment of 2 with similar results. To ascertain antigen-presenting function of MCL cells, FC-muMCL1 cells (1 105 cells/well) were treated with LPS (2 mg/mL), LPS þ increasing concentrations of CPA-7, or left untreated (media) for 24 hours. Then, cells were washed 4 þ and plated with either 5 10 naive or anergized anti-OVA CD4 T cells isolated from tumor-bearing mice in presence of 3 mg/mL of OVA peptide323–339. Forty-eight hours later, supernatants were collected and the production of IL-2 and IFN- by naive T cells (C) and the production of IFN-g by anergized T cells (D) were determined by ELISA. E, expression of B7.1 and B7.2 by FC-muMCL1 cells treated or not treated with CPA-7 (15 mmol) for 15 hours was assessed by flow cytometry. Gray histogram shows isotype control. Shown is a representative experiment of 3 independent experiments with similar results (, P < 0.05 statistically significant for the difference in cytokine production).

þ cells and restoration of responsiveness of tolerized CD4 T us to determine the in vivo anti-MCL effect of CPA-7 in a larger cells. group of animals. C57BL/6 mice were challenged with 5 106 FC-muMCL1 cells given subcutaneously. Half of the mice In vivo inhibition of Stat3 delays progression of murine received vehicle control and the other half received CPA-7 B-cell lymphomas (5 mg/kg/i.v. every 3 days, starting on day þ5 after tumor Next, we determined whether CPA-7 inhibits Stat3 signaling challenge). Unlike untreated MCL-bearing mice, which rapidly in malignant B cells in vivo. Previous studies have shown that developed tumors (Fig. 5B, solid line), mice treated with CPA-7 CPA-7 induces in vivo antitumor responses when used at the had a significant delay in MCL tumor growth (Fig. 5B, dashed dose of 5 mg/kg given i.v. every 3 days (16). We therefore line). Of note, analysis of immune cells infiltrating MCL lym- injected 5 106 FC-muMCL1 cells i.p. into C57BL/6 mice and phoma nodules revealed no differences in intratumoral T-cell 21 days later, animals were treated (or not) with 5 mg/kg of recruitment (Supplementary Fig. S2). Similarly, BALB/c mice CPA-7 given i.v. on days þ21, þ24, and þ27. Two days later (day challenged with 1 106 A20 lymphoma cells subcutaneously þ29), animals were sacrificed and tumor nodules were dis- and then treated with CPA-7 (same dose and frequency as in sected from their livers. As shown in Fig. 5A, a decrease in the MCL model) rejected this B-cell tumor (Fig. 5C, dashed phospho-Stat3 expression was observed in malignant B cells line). No such rejection was observed in A20 B cell lymphoma- isolated from MCL-bearing mice treated with CPA-7. No such bearing mice given vehicle control (Fig. 5C, solid line). There- effect was observed in untreated tumor-bearing mice or ani- fore, in vivo treatment of lymphoma-bearing mice with CPA-7 mals treated with vehicle control. Of note, less tumor burden resulted in decreased Stat3 phosphorylation in malignant B was observed in CPA-7–treated mice. These results prompted cells and a strong anti-lymphoma effect.

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Figure 5. In vivo treatment with CPA-7 results in decreased Stat3 phosphorylation in malignant B cells and an anti-lymphoma effect. A, 5 106 FC-muMCL1 cells were injected i.p. into C57BL/6 mice. Twenty-one days later, animals were treated or not treated with 5 mg/kg of CPA-7 given i.v. every 3 days (days þ21, þ24, and þ27). On day þ29, animals were sacriﬁced and tumor nodules were carefully dissected from their livers. Malignant B cells were then isolated and the expression of phopho-Stat3 was determined by Western blot analysis using an anti-p-Stat3 (Tyr705) antibody. Shown is a representative of 2 experiments with similar results. B, C57BL/6 mice (n ¼ 10) were challenged with 5 106 FC-muMCL1 cells given subcutaneously in the right leg. Half the mice were then treated with CPA-7 (5 mg/kg) given i.v. every 3 days, starting on day þ5 after tumor challenge (dashed line). The other half of the mice received vehicle control (solid line). Tumor volumes were calculated (length width width 1/2) from measurements as indicated. Two independent experiments were carried with similar results. C, BALB/c mice (n ¼ 10) were challenged with 1 106 A20 lymphoma cells given subcutaneously in the right leg. Half the mice were treated with CPA-7 (dash line) and the other half with vehicle control (solid line) as indicated in B. Animals were monitored for the development of tumor and tumor volumes measured on days indicated. Two independent experiments were carried out with similar results (P < 0.01).

The in vivo antitumor effect of CPA-7 requires an intact treated with CPA-7 in which a strong antitumor effect was adaptive immune system clearly shown (Fig. 5B and C), such an antitumor effect was not Previous studies have shown that disruption of Stat3 in observed in immunodeﬁcient animals treated with CPA-7. malignant cells, including MCL cells, resulted in the induction Indeed, no difference in the kinetics of tumor growth was of apoptosis (27, 28). As such, the in vivo antitumor effect observed among untreated or CPA-7–treated lymphoma-bear- observed in CPA-7–treated lymphoma-bearing mice (Fig. 5) ing SCID mice (Fig. 6A and B, MCL and A20, respectively). To could be a reﬂection of a direct effect of this drug upon tu- rule out the possibility that CPA-7 might not be affecting its mor cells themselves rather than immune effects triggered by tumor target in SCID mice, we determined the expression of p- Stat3 inhibition. To address this question, C57BL/6 SCID mice STAT3 in malignant B cells isolated from tumor nodules of (Fig. 6A) or BALB/c-SCID mice (Fig. 6B) were challenged with untreated and CPA-7–treated SCID mice. As shown in Fig. 6C, 5 106 FC-muMCL1 cells or 1 106 A20 lymphoma cells given in vivo treatment with CPA-7 resulted in decreased p-Stat3 subcutaneously, respectively. Then, half the mice in each group expression, an effect that was more pronounced in A20 lym- were treated with CPA-7 (5 mg/kg every 3 days, starting on day phoma cells. These results indicate that the antitumor effect of þ5 after tumor challenge) and the other half received vehicle CPA-7 requires an intact adaptive immune system and points control. Unlike immunocompetent lymphoma-bearing mice to the immunologic rather than the nonimmunologic

Figure 6. Similar kinetics of lymphoma growth in CPA-7 treated SCID mice. A, C57BL/6 SCID mice (n ¼ 10) were challenged with 5 106 Fc-muMCL1 cells given subcutaneously. Then, mice were treated with CPA-7 (dash line; 5 mg/kg i.v. given every 3 days, starting on day þ5 after tumor challenge) or vehicle control (solid line). B, BALB/c SCID mice (n ¼ 10) were challenged with 1 106 A20 cells given subcutaneously. Mice were treated with CPA-7 (dash line) or vehicle control (solid line) as indicated in A. Tumors were measured at the indicated times. C, C57BL/6 SCID and BALB/c SCID mice were treated as indicated in A and B. Mice were sacriﬁced 19 days after tumor challenge and tumor nodules were collected. Malignant B cells were then isolated and the expression of phopho-Stat3 was determined by Western blot analysis using an anti-p-Stat3 (Tyr705) antibody. Two independent experiments were carried out with similar results.

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In the in vivo immune response against B-cell lymphomas, it is likely that both malignant cells themselves as well as bone marrow–derived APCs present tumor antigens to antigen- þ specific CD4 T cells. B-cell lymphomas are the transformed counterparts of cells endowed with antigen-presenting capabilities. Normal B lymphocytes have long been known to þ interact with CD4 T cells during physiologic immune responses in a process that involves presentation of peptide MHC class II complexes, along with costimulatory signals to antigen-specific T cells (31, 32). Like normal B cells, malignant B cells also express MHC class I and II molecules and low but inducible levels of adhesion and costimulatory molecules (1, 33, 34). In spite of these intrinsic properties, it is quite paradoxical that B-cell malignancies fail to be eliminated in the very same compartment lymph nodes where tumor antigen– specific T-cell responses are initiated. Several factors might account for the failure of malignant B cells to properly activate T cells in vivo. First, their expression of MHC molecules, costimulatory molecules, and/or adhesion Figure 7. CPA-7 specifically inhibits Stat3 phosphorylation in human MCL molecules that participate in T-cell priming might not be cells. JEKO cells were treated or not treated with CPA-7 (30 mmol) for sufficient to trigger full T-cell activation. This "state" of partial 24 hours (left). In parallel, cells were transiently transfected with Stat3- T-cell activation, in the absence of additional signals capable of specific siRNA (Stat3 siRNA) or nontargeting control (Control). Then, cells were harvested and protein extracts were obtained and subjected to sustaining this initial response and/or in the presence of Western blot analysis using antibodies against p-Stat3, Stat3, p-MAPK, dominant suppressive mechanisms, might be followed instead MAPK, p-Akt, and Akt. Shown is a representative experiment of 2 with by a state of T-cell anergy (1, 35). Among the active suppressive similar results. mechanisms, the ability of malignant B cells to induce T-cell immunologic synapse dysfunction (36) together with the ability antitumor effects of this Stat3 inhibitor, as playing a dominant of bone marrow–derived APCs to create a tolerogenic envi- in vivo role in its anti-lymphoma activity. ronment that favors T-cell anergy over T-cell activation (37–39) have gained particular attention. It is plausible therefore that CPA-7 inhibits Stat3 in human MCL the combination of these mechanisms would be conducive to Next, we determined whether CPA-7 would also inhibit Stat3 T-cell unresponsiveness, a barrier that needs to be overcome if in human MCL cells. As shown in Fig. 7, p-Stat3 was diminished effective immunity against B-cell tumors is to be generated. in JEKO cells treated in vitro with CPA-7. This inhibition was In the past several years, a number of therapeutic specific as other signaling pathways such as phospho-MAPK or approaches have sought to improve the antigen-presenting phospho-AKT were not affected in MCL cells treated with even capabilities of malignant B cells by either genetically modifying higher doses of CPA-7 (30 mmol). The selectivity of this agent is these cells to enforce the expression of adhesion and costi- further highlighted by the demonstration that the phenotype mulatory molecules as well as proinflammatory cytokines (40– displayed by CPA-7–treated JEKO cells is recapitulated in cells 43), or by repairing a dysfunctional T-cell immunologic syn- in which Stat3 was knocked down using specific Stat3 siRNA apse with immunomodulatory drugs such as lenalidomide (36). (Fig. 7, right). Of note, in vitro culture of CPA-7–treated human Other approaches have focused instead on the induction of JEKO cells with allogeneic human peripheral blood mononu- inflammatory APCs as a strategy to improve cross-presenta- clear cells also resulted in enhanced IL-2 and IFN-g production tion of tumor antigens to antigen-specific T cells (3, 44). by T cells as determined by ELISPOT assay (data not shown). Although each of these approaches induced productive immune responses, the duration and magnitude of these Discussion effects were transient and not strong enough to fully eradicate In this study, we have shown that genetic or pharmacologic systemic lymphoma. A potential explanation for their limited disruption of Stat3 in malignant B cells increased their immu- success is that they have targeted either the malignant B cell or þ nogenicity leading to augmentation of antigen-specific CD4 the APC, but not both, and as such, they were unable to fully T-cell function and restoration of responsiveness of tolerized T overcome tolerogenic mechanisms in cancer. Therefore, from a cells. These findings expand the previously known proinflam- therapeutic perspective, it would be desirable to find novel matory effects of Stat3 inhibition upon other immune cells approaches with the dual ability of enhancing the antigen- such as bone marrow–derived APCs (17, 29, 30). This unique presenting function of malignant B cells and inducing inflam- property of Stat3 inhibition to influence the inflammatory matory APCs displaying enhanced cross-presentation of tumor status of both the malignant B cell as well as the APC points antigens to antigen-specific T cells. to pharmacologic inhibition of this signaling pathway as an Inhibition of Stat3 signaling represents a novel strategy given appealing strategy to overcome tolerance to tumor antigens its known ability to influence the inflammatory status of the and elicit a strong antitumor immunity. APC (17, 29, 45) and, as shown here, to augment the APC

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Cheng et al.

function of malignant B cells. Indeed, treatment of malignant B tumor immune responses also triggered by the effects of Stat3 cells with CPA-7 rendered these cells better activators of inhibition upon APCs and other immune cells (26, 48). This þ antigen-specific CD4 T cells and capable of restoring the proinflammatory environment generated by Stat3 inhibition is responsiveness of tolerant T cells isolated from lymphoma- further enhanced by the inability of malignant B cells and bearing mice. In addition to these in vitro effects, treatment of immune cells to produce IL-10 in the absence of intact Stat3 MCL-bearing mice with CPA-7 decreased Stat3 phosphoryla- signaling (15, 17). Such a lack of production of IL-10 has the tion in tumor cells and resulted in protective immunity. Of note, dual advantage of not only diminishing the generation of an the lack of antitumor activity in immunodeficient mice treated immunosuppressive environment but also depriving malig- with CPA-7 points to the effects of Stat3 inhibition upon nant B cells of an important survival factor (49, 50). immune cells as being essential for effective lymphoma erad- Taken together, the dual effects of Stat3 inhibition upon ication in vivo. It should be mentioned that our in vivo results in both the malignant B cells as well as immune cells triggers a SCID mice are at odds with the results reported by Wang and positive loop of proinflammatory events that likely generates colleagues who treated SCID mice bearing human SP53 or an activating rather than a tolerogenic environment in the Grant 519 MCL cells with Atiprimod, a compound known to lymph nodes, which might be ultimately conducive to effective inhibit Stat3 (27). Unlike our study, SCID mice treated with antilymphoma immunity. Such a unique property of Stat3 Atipromid had significantly less tumor burden compared with inhibition makes this approach suitable for future evaluation control mice, pointing to a direct antitumor effect of this in human MCL and other B-cell malignancies, either alone or in compound upon MCL cells. Several differences between their combination with lenalidomide, or as an adjuvant to thera- study and ours might explain these seemingly divergent out- peutic vaccines. comes. First, unlike CPA-7, which mainly inhibits Stat3 without affecting other signaling pathways in human MCL (Fig. 7), Disclosure of Potential Conflicts of Interest Atipromid is a less selective inhibitor of Stat3 as it also activates No potential conflicts of interest were disclosed. c-jun-NH2-kinase and inhibits NF-kB, an important survival pathway that is constituvely activated in MCL. It is plausible, Authors' Contributions Conception and design: H. Wang, P. Horna, S. Sebti, J. Tao, E.M. Sotomayor therefore, that the antitumor effect observed in their study Development of methodology: F. Cheng, H. Wang, A. Villagra, M. Smith, E.M. could be related to the targeting of pathway(s) other than Stat3. Sotomayor Second, in our in vivo studies, mice were challenged with Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Wang, M. Smith murine MCL cells, whereas in their study, SCID mice were chal- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, lenged with human MCL cells. Third, there were also differ- computational analysis): F. Cheng, H. Wang, A. Villagra, J. Pinilla-Ibarz, J. Tao, in vivo E.M. Sotomayor ences in the dose and frequency of administration of Writing, review, and/or revision of the manuscript: H. Wang, P. Horna, B. CPA-7 and Atipromid (CPA-7: 5 mg/kg/i.v. every 3 days, starting Shah, K.V. Woan, A. Villagra, J. Pinilla-Ibarz, S. Sebti, M. Smith, E.M. Sotomayor on day þ5 after tumor challenge vs. Atipromid: 25 mg/kg/i.p. Administrative, technical, or material support (i.e., reporting or orga- – nizing data, constructing databases): H. Wang, Z. Wang, E. Sahakian daily for 6 days starting 3 4 weeks after tumor challenge). Study supervision: A. Villagra, J. Pinilla-Ibarz, E.M. Sotomayor Our demonstration that Stat3 inhibition is an effective strategy in a murine model of MCL provides the framework Acknowledgments for its future combination with agents able to repair defective The authors thank Moffitt's Flow Cytometry Core and Animal Facility for T-cell immunologic synapse, such as lenalidomide, or as technical assistance. adjuvants to lymphoma vaccines. Furthermore, our findings, together with the demonstration that Stat3 is constitutively Grant Support This work was supported by PHS grants CA134807 and CA087583 (E.M. activated in human MCL cells (46, 47), provide the basis for Sotomayor). evaluating the clinical efficacy of Stat3 inhibition in human The costs of publication of this article were defrayed in part by the payment of MCL. Of note, inhibition of Stat3 in tumor cells displaying page charges. This article must therefore be hereby marked advertisement in aberrant activation of this pathway has been shown to result in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. fl "in ammatory death", a process associated with release of Received November 4, 2011; revised June 13, 2012; accepted June 13, 2012; proinflammatory mediators that could amplify ongoing anti- published OnlineFirst June 22, 2012.

References 1. Sotomayor EM, Borrello I, Rattis FM, Cuenca AG, Abrams J, Staveley- 4. Howard OM, Gribben JG, Neuberg DS, Grossbard M, Poor C, Janicek O'Carroll K, et al. Cross-presentation of tumor antigens by bone MJ, et al. Rituximab and CHOP induction therapy for newly diagnosed marrow-derived antigen-presenting cells is the dominant mechanism mantle-cell lymphoma: molecular complete responses are not predic- in the induction of T-cell tolerance during B-cell lymphoma progres- tive of progression-free survival. J Clin Oncol 2002;20:1288–94. sion. Blood 2001;98:1070–7. 5. Witzig TE. Current treatment approaches for mantle-cell lymphoma. J 2. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive Clin Oncol 2005;23:6409–14. strategies that are mediated by tumor cells. Annu Rev Immunol 6. Martin P, Chadburn A, Christos P, Weil K, Furman RR, Ruan J, et al. 2007;25:267–96. Outcome of deferred initial therapy in mantle-cell lymphoma. J Clin 3. Horna P, Cuenca A, Cheng F, Brayer J, Wang HW, Borrello I, et al. In Oncol 2009;27:1209–13. vivo disruption of tolerogenic cross-presentation mechanisms 7. Fuchs EJ, Bedi A, Jones RJ, Hess AD. Cytotoxic T cells overcome uncovers an effective T-cell activation by B-cell lymphomas leading BCR-ABL-mediated resistance to apoptosis. Cancer Res 1995;55: to antitumor immunity. Blood 2006;107:2871–8. 463–6.

OF8 Cancer Res; 72(17) September 1, 2012 Cancer Research

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8. Shtil AA, Turner JG, Durfee J, Dalton WS, Yu H. Cytokine-based tumor 30. Nefedova Y, Nagaraj S, Rosenbauer A, Muro-Cacho C, Sebti SM, cell vaccine is equally effective against parental and isogenic multi- Gabrilovich DI. Regulation of dendritic cell differentiation and antitumor drug-resistant myeloma cells: the role of cytotoxic T lymphocytes. immune response in cancer by pharmacologic-selective inhibition of Blood 1999;93:1831–7. the janus-activated kinase 2/signal transducers and activators of 9. Neelapu SS, Kwak LW, Kobrin CB, Reynolds CW, Janik JE, Dunleavy transcription 3 pathway. Cancer Res 2005;65:9525–35. K, et al. Vaccine-induced tumor-specific immunity despite severe B- 31. Clark EA, Ledbetter JA. How B and T cells talk to each other. Nature cell depletion in mantle cell lymphoma. Nat Med 2005;11:986–91. 1994;367:425–8. 10. Kaufmann H, Raderer M, Wohrer€ S, Pusp€ ok€ A, Bankier A, Zielinski C, 32. Attanavanich K, Kearney JF. Marginal zone, but not follicular B cells, et al. Antitumor activity of rituximab plus thalidomide in patients with are potent activators of naive CD4 T cells. J Immunol 2004;172:803–11. relapsed/refractory mantle cell lymphoma. Blood 2004;104:2269–71. 33. Chaperot L, Plumas J, Jacob MC, Bost F, Molens JP, Sotto JJ, et al. 11. Habermann TM, Lossos IS, Justice G, Vose JM, Wiernik PH, McBride Functional expression of CD80 and CD86 allows immunogenicity of K, et al. Lenalidomide oral monotherapy produces a high response rate malignant B cells from non-Hodgkin's lymphomas. Exp Hematol in patients with relapsed or refractory mantle cell lymphoma. Br J 1999;27:479–88. Haematol 2009;145:344–9. 34. Guilloux Y, Bai XF, Liu X, Zheng P, Liu Y. Optimal induction of effector 12. Zhou Y, Zhang L, Romaguera J, Delasalle K, Han X, Du X, et al. but not memory antitumor cytotoxic T lymphocytes involves direct Immunotherapy in mantle cell lymphoma: anti-CD20-based therapy antigen presentation by the tumor cells. Cancer Res 2001;61:1107–12. and beyond. Am J Hematol 2008;83:144–9. 35. Adler AJ, Marsh DW, Yochum GS, Guzzo JL, Nigam A, Nelson WG, 13. Darnell JE Jr. STATs and gene regulation. Science 1997;277:1630–5. et al. CD4(þ) T cell tolerance to parenchymal self-antigens requires 14. Sano S, Itami S, Takeda K, Tarutani M, Yamaquchi Y, Miura H, et al. presentation by bone marrow-derived antigen-presenting cells. J Exp Keratinocyte-specific ablation of Stat3 exhibits impaired skin remodel- Med 1998;187:1555–64. ing, butdoes not affect skin morphogenesis. EMBO J 1999;18:4657–68. 36. Ramsay AG, Clear AJ, Kelly G, Fatah R, Matthews J, MacDougall F, et al. 15. Takeda K, Clausen BE, Kaisho T, Tusjimura T, Terada N, Forster€ I, et al. Follicular lymphoma cells induce T-cell immunologic synapse dysfunc- EnhancedTh1 activityand development ofchronic enterocholitis inmice tion that can be repaired with lenalidomide: implication for the tumor devoid of Stat3 in macrophages and neutrophils. Immunity 1999;10:39. microenvironment and immunotherapy. Blood 2009;114:4713–20. 16. Kortylewski M, Jove R, Yu H. Targeting STAT3 affects melanoma on 37. Dave SS, Wright G, Tan B, Rosenwald A, Gascoyne RD, Chan WC, multiple fronts. Cancer Metastasis Rev 2005;24:315–27. et al. Prediction of survival in follicular lymphoma based on molecular 17. Cheng F, Wang HW, Cuenca A, Huang M, Ghansah T, Brayer J, et al. A features of tumor-infiltrating immune cells. N Engl J Med 2004;351: critical role for Stat3 signaling in immune tolerance. Immunity 2003; 2159–69. 19:425–36. 38. Zhou G, Lu Z, McCadden JD, Levitsky HI, Marson AL, Reciprocal 18. Kirberg J, Baron A, Jakob S, Rolink A, Karjalainen K, von Boehmer H. changes in tumor antigenicity and antigen-specific T cell function Thymic selection of CD8þ single positive cells with a class II major histo- during tumor progression. J Exp Med 2004;200:1581–92. compatibility complex-restricted receptor. J Exp Med 1994;180:25–34. 39. Haabeth OA, Lorvik KB, Hammarstrom C, Donaldson IM, Haraldsen G, 19. Barnden MJ, Allison J, Heath WR, Carbone FR. Defective TCR expres- Bogen B, et al. Inflammation driven by tumour-specific Th1 cells sion in transgenic mice constructed using cDNA-based alpha- and protects against B-cell cancer. Nat Commun 2011;2:240. beta-chain genes under the control of heterologous regulatory ele- 40. Levitsky HI, Montgomery J, Ahmadzadeh M, Staveley-O'Carroll K, ments. Immunol Cell Biol 1998;76:34–40. Guarnieri F, Longo DL, et al. Immunization with granulocyte-macro- 20. Staveley-O' Carroll K, Sotomayor E, Montgomery J, Borrello I, Hwang phage colony-stimulating factor- transduced, but not B7-1-trans- L, Fein S, et al. Induction of antigen-specific T cell anergy: an early duced, lymphoma cells primes idiotype- specific T cells and generates event in the course of tumor progression. Proc Natl Acad Sci U S A potent systemic antitumor immunity. J Immunol 1996;156:3858–65. 1998;95:1178–83. 41. Maric M, Chen L, Sherry B, Liu Y. A mechanism for selective recruitment 21. Smith MR, Joshi I, Jin F, Al-Saleem T. Murine model for mantle cell of CD8 T cells into B7-1-transfected plasmacytoma: role of macro- lymphoma. Leukemia 2006;20:891–3. phage-inflammatory protein 1alpha. J Immunol 1997;159:360–8. 22. Catlett-Falcone R, Landowski TH, Oshiro MM, Turkson J, Levitzki A, 42. Cantwell MJ, Wierda WG, Lossos IS, Levy R, Kipps TJ. T cell activation Savino R, et al. Constitutive activation of Stat3 signaling confers following infection of primary follicle center lymphoma B cells with resistance to apoptosis in human U266 myeloma cells. Immunity adenovirus encoding CD154. Leukemia 2001;15:1451–7. 1999;10:105–15. 43. Briones J, Timmerman J, Levy R. In vivo antitumor effect of CD40L- 23. Niu G, Heller R, Catlett-Falcone R, Coppola D, Jaroszeski M, Dalton W, transduced tumor cells as a vaccine for B-cell lymphoma. Cancer Res et al. Gene therapy with dominant-negative Stat3 suppresses growth 2002;62:3195–9. of the murine melanoma B16 tumor in vivo. Cancer Res 1999;59: 44. Evel-Kabler K, Song XT, Aldrich M, Huang XF, Chen SY. SOCS1 5059–63. restricts dendritic cells' ability to break self tolerance and induce 24. Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y, Pestell RG, antitumor immunity by regulating IL-12 production and signaling. J Albanese C, et al. Stat3 as an oncogene. Cell 1999;98:295–303. Clin Invest 2006;116:90–100. 25. Turkson J, Kim JS, Zhang S, Yuan J, Huang M, Glenn M, et al. Novel 45. Brayer J, Cheng F, Wang H, Horna P, Vicente-Suarez I, Pinilla-Ibarz J, peptidomimetic inhibitors of signal transducer and activator of transcrip- et al. Enhanced CD8 T cell cross-presentation by macrophages with tion3dimerizationandbiologicalactivity.MolCancerTher2004;3:261–9. targeted disruption of STAT3. Immunol Lett 2010;131:126–30. 26. Kortylewski M, Kujawski M, Wang T, Wei S, Zhang S, Pilon-Thomas S, 46. Lai R, Rassidakis GZ, Medeiros LJ, Leventaki V, Keating M, McDonnell et al. Inhibiting Stat3 signaling in the hematopoietic system elicits TJ. Expression of STAT3 and its phosphorylated forms in mantle cell multicomponent antitumor immunity. Nat Med 2005;11:1314–21. lymphoma cell lines and tumours. J Pathol 2003;199:84–9. 27. Wang M, Zhang L, Han Xiaohong, Yang Jing, Qian Jianfei, Hong 47. Baran-Marszak F, Boukhiar M, Harel S, Laquiller C, Roger C, Gressin R, Sungyoul, et al. Atiprimod inhibits the growth of mantel cell lymphoma et al. Constitutive and B-cell receptor-induced activation of STAT3 are in vitro and in vivo and induces apoptosis via activating the mitochon- important signaling pathways targeted by bortezomib in leukemic drial pathway. Blood 2007;109:5455–62. mantle cell lymphoma. Haematologica 2010;95:1865–72. 28. Niu G, Shain KH, Huang M, Ravi R, Bedi A, Dalton WS, et al. Over- 48. Yu H, Jove R. The STATs of cancer–new molecular targets come of expression of a dominant-negative signal transducer and activator of age. Nat Rev Cancer 2004;4:97–105. transcription 3 variant in tumor cells leads to production of soluble 49. Madan R, Demircik F, Surianarayanan S, Allen JL, Divanovic S, Tromp- factors that induce apoptosis and cell cycle arrest. Cancer Res ette A, et al. Nonredundant roles for B cell-derived IL-10 in immune 2001;61:3276–80. counter-regulation. J Immunol 2009;183:2312–20. 29. Nefedova Y, Cheng P, Gilkes D, Blaskovich M, Beg AA, Sebti SM, et al. 50. Scuto A, Kujawski M, Kowolik C, Krymskaya L, Wang L, Weiss LM, Activation of dendritic cells via inhibition of Jak2/STAT3 signaling. J et al. STAT3 inhibition is a therapeutic strategy for ABC-like diffuse Immunol 2005;175:4338–46. large B-cell lymphoma. Cancer Res 2011;71:3182–8.

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Stat3 Inhibition Augments the Immunogenicity of B-cell Lymphoma Cells, Leading to Effective Antitumor Immunity

Fengdong Cheng, Hongwei Wang, Pedro Horna, et al.

Cancer Res Published OnlineFirst June 22, 2012.

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