STAT3 Blockade Research Articles

Abstract A21: The PRC1 protein RING1B contributes to Ewing sarcoma tumorigenesis by blocking the NaV1.6 sodium channel and modulating the NF-κB pathway, independently of the fusion oncoprotein

Abstract Objective: Ewing sarcoma (ES) is an aggressive and poorly differentiated tumor, commonly arising in adolescents and young adults. It is molecularly defined by a reciprocal translocation resulting in the fusion of EWS to an ETS transcription factor, being EWS-FLI the most common chimera. EWS-FLI1 up-regulates gene expression through the interaction with GGAA repeats, however, data obtained by depleting EWS-FLI1 revealed that more genes are down-regulated than up-regulated. A variety of repressive mechanisms have been described including the polycomb group (PcG) of proteins EZH2 and BMI1 that contribute to the epigenetic repressive landscape in ES. We have characterized the expression and function of the PcG RING1B in ES. Methods: 18 ES primary tissues were evaluated. The intensity of the immunostaining was evaluated and scored for the PcG RING1B, EZH2, MEL18 and BMI1. Western blot quantification was performed using Image J densitometry software. RNA was analyzed by qRT-PCR assays using SYBR Green PCR master mix from Applied Biosystems. ES cell lines (A4573, A673, SK-ES-1, TC71) were grown in standard conditions and treated with DNZep, TSA, SU5402, NSC87877, S3I-201 and BMS-345541 for 24 hours. Mock, RING1B, BMI1, FLI1 and EZH2 shRNA and siRNAs were generated using standard methods. Microarray analysis was performed according to protocols from Ambion and Affymetrix, and hybridized to GeneChip Human Gene 2.0 ST Array (Affymetrix). Each experiment was performed in triplicate at least three times. Statistical analysis was performed using the Student's t-test with a p < 0.05 as significant. Results: RING1B was highly and universally expressed in all primary ES tumor specimens. In ES cell lines, RING1B mRNA levels were not affected by oncogene down-regulation or treatment with differentiation agents (ATRA; BHA). When RING1B was depleted in ES cell lines by retroviral shRNA, heme biosynthesis and hematopoiesis were 2 of the canonical pathways identified and the set of genes regulated by RING1B unique. Among the most up-regulated genes upon RING1B depletion stand FGF14 and SCN8A. Repression of the 2 components of the NaV1.6 channel was proved specific for RING1B and functionally relevant in ES. Upon RING1B depletion, NF-κB was the most significantly altered pathway identified. Expression of the NF-κB protein p105 (and p50) was found enhanced and RING1B depletion sensitized specifically ES cells to apoptosis by FGFR-SHP2 and STAT3 blockade. Conclusion: we show that RING1B is pivotal in ES cells, independent of the fusion oncogene, likely reflecting traits of the cell of origin. Citation Format: Inmaculada Hernandez-Muñoz, Elisabeth Figuerola, Sara Sanchez-Molina, Jaume Mora. The PRC1 protein RING1B contributes to Ewing sarcoma tumorigenesis by blocking the NaV1.6 sodium channel and modulating the NF-κB pathway, independently of the fusion oncoprotein. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A21.

Abstract A12: Microenvironmental Interleukin-6 induced activation of STAT3 signaling in medulloblastoma: Implications for molecular pathology and therapy

Abstract Aberrant activation of Janus kinase (JAK) / signal transducer and activator of transcription (STAT) 3 pathway has been implicated in tumor initiation, progression and metastasis which make it a potential therapeutic target in human malignancies. In vitro STAT3 inhibition exhibited anti-tumor effect in medulloblastoma (MB) cell lines; however, little has been reported about the mechanisms of STAT3 activation in medulloblastoma (MB). We found that STAT3 was constitutively activated in both medulloblastoma patient samples and 3 mice models that were clinically classified as SHH subgroup, Group 3 and Group 4 in pediatric MB patients. By using PCR arrays we investigated the early expression profile of local cytokines in host cerebellum during MB pathogenesis and found increased IL17B, IL6 and TNFα in the microenvironment of MB mice models. In vitro cytokine stimulation assay indicated that IL6 was the major activator of STAT3 in D283 MB cells. Confocal immunofluorescence staining of different cell components in the cerebellum in D283 xenografts showed that the endothelial cells and Purkinje cells were the two major sources of IL6 secretion. Our data provides molecular and cellular clue for the activation of STAT3 signaling during MB tumorigenesis and might help to develop more effective therapeutic targets for STAT3 blockade in MB. Citation Format: Shuang Yan, Sampurna Chatterjee, Trupti Vardam, Shuji Kitahara, Tai Hato, Sylvie Roberge, Vasileios Askoxylakis, Mark Duquette, Dai Fukumura, Lei Xu, Rakesh Jain. Microenvironmental Interleukin-6 induced activation of STAT3 signaling in medulloblastoma: Implications for molecular pathology and therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A12.

Nuclear translocation of STAT3 by in vitro metreleptin administration causes lipolysis in human primary adipocytes

We utilized subcutaneous (SC)- and omental (OM)-derived human primary adipocytes (hPA) from obese male, and investigated whether synthetic analog of leptin, metreleptin, may regulate lipolysis via translocation of STAT3 to the nucleus. We observed that 50 ng/mL of metreleptin increases STAT3 phosphorylation in both SC- and OM-derived hPA. Importantly, we found for the first time that metreleptin is capable of trans-locating STAT3 to the nucleus and STAT3 blockade inhibits metreleptin-induced lipolysis. Our initial data provide novel insights into the role of STAT3 as probable mediator of the action of metreleptin in regulating metabolism.

STAT3 Blockade Inhibits Radiation-Induced Malignant Progression in Glioma.

High grade gliomas (HGG) are classified into four subgroups based on transcriptional signatures and phenotypic characteristics. In particular, the proneural-to-mesenchymal transition (PMT) is associated with increased malignancy, poor prognosis, and disease recurrence, but the underlying causes of PMT are still unclear. In this study, we investigated whether radiotherapy promotes PMT using a genetically engineered mouse model of proneural HGG. We found that cranial ionizing radiation induced robust and durable PMT in tumors. Additionally, we isolated primary proneural HGG cells from mouse and human tumors and demonstrate that radiation induced a sustained cell-intrinsic mesenchymal transition associated with increased invasiveness and resistance to the alkylating agent temozolomide. Expectedly, irradiation-induced PMT was also associated with activation of the STAT3 transcription factor, and the combination of STAT3 blockade using JAK2 inhibitors with radiation abrogated the mesenchymal transition and extended survival of mice. Taken together, our data suggest that clinical JAK2 inhibitors should be tested in conjunction with radiation in patients with proneural HGG as a new strategy for blocking the emergence of therapy-resistant mesenchymal tumors at relapse.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Loss-of-Function PTPRD Mutations Lead to Increased STAT3 Activation and Sensitivity to STAT3 Inhibition in Head and Neck Cancer.

BackgroundProtein tyrosine phosphatase receptor type D (PTPRD) is a putative tumor suppressor in several cancers including head and neck squamous cell carcinoma (HNSCC). STAT3 is a frequently hyperactivated oncogene in HNSCC. As STAT3 is a direct substrate of PTPRD, we sought to determine the genetic or epigenetic alterations of PTPRD that contribute to overactive STAT3 in HNSCC.MethodsWe analyzed data from The Cancer Genome Atlas (TCGA) and our previous whole-exome sequencing study and summarized the mutation, methylation, and copy number status of PTPRD in HNSCC and other cancers. In vitro studies involved standard transfection and MTT protocols, as well as methylation-specific PCR.ResultsOur findings indicate that PTPRD mutation, rather than methylation or copy number alteration, is the primary mechanism by which PTPRD function is lost in HNSCC. We demonstrate that overexpression of wild-type PTPRD in HNSCC cells significantly inhibits growth and STAT3 activation while PTPRD mutants do not, suggesting that mutation may lead to loss of function and subsequent hyper-phosphorylation of PTPRD substrates, especially STAT3. Importantly, we determined that HNSCC cells harboring an endogenous PTPRD mutation are more sensitive to STAT3 blockade than PTPRD wild-type cells. We additionally found that PTPRD mRNA expression does not correlate with pSTAT3 expression, suggesting that alterations that manifest through altered mRNA expression, including hypermethylation and gene copy number alterations, do not significantly contribute to STAT3 overactivation in HNSCC.Conclusion PTPRD mutation, but not methylation or copy number loss, may serve as a predictive biomarker of sensitivity to STAT3 inhibitors in HNSCC.

Abstract 1346: Immunotherapy against breast cancer based on Stat3 blockade

Abstract The signal transducer and activator of transcription 3 (Stat3) is constitutively active in breast cancer (BC) and contributes to malignant transformation by promoting cell cycle progression, inhibiting apoptosis and mediating tumor immune evasion. Stat3 inhibition induces expression of proinflammatory cytokines and chemokines. On the other hand, it has been shown that oncogene inactivation induces cellular senescence and the secretion of senescence-associated secretome (SAS) in diverse tumor types. We recently described that Stat3 blockade leads to a senescence program in murine BC cells, which is accompanied by the secretion of proinflammatory cytokines. In addition, we demonstrated that immunization of mice with syngeneic Stat3 blocked BC cells induces an antitumoral immune response that involves the participation of CD4+ Th cells and cytotoxic NK cells. In this study our objectives were to study whether immunization with supernatants (SN) produced by Stat3-blocked cells induces an antitumor immune response and to explore the contribution of senescence phenotype in this response. For that purpose we used BC models of ErbB-2-positive, JIMT-1 and KPL-4 cells (human) and C4HD cells (murine), and of triple negative, MDA-MB231 cells (human) and 4T1 cell (murine). Knockdown of Stat3 with siRNA in these cells, induced senescence (assessed by acidic β-galactosidase staining). In human BC cells the senescent phenotype was accompanied by up-regulation of p21cip1 and downregulation of Rb expressions. In mouse BC cells we observed an increased expression of p16ink4a. In addition, simultaneous transfection with siRNAs targeting Stat3 and p16ink4a reverted the senescent phenotype. Then, we used a pre-clinical model in which we embedded the SN of C4HD cells in a slow-delivery depot as an adjuvant of a cellular immunotherapy. We immunized the animals with irradiated C4HD cells together with a depot containing lyophilized SN of C4HD cells transfected in vitro either with Stat3 siRNA (senescent) and Stat3 and p16ink4a siRNA (non-senescent), or a control siRNA. After 3 immunizations, with 15 d of interval between them, animals were challenged with C4HD tumor and tumor growth was monitored for 40 d. We observed that immunization with SN of cells with Stat3 siRNA decreased tumor growth vs. control siRNA group. Interestingly, there was no difference between tumor growth of animals immunized with SN of cells with Stat3 siRNA vs Stat3 and p16ink4a siRNA. We observed in these two groups greater cytotoxic activity of NK cells and an increase in the number of memory CD4+ T cells vs. control. These results suggest that Stat3 blockade drives a senescence program also in human BC cells. The secretome of Stat3-blocked BC cells is an effective adjuvant and could be formulated for immunotherapies independently of the SAS. Defining the protein, peptide, cytokine, chemokine composition of this supernatant opens up new avenues for immunotherapy in cancer patients. Citation Format: Mara De Martino, María Florencia Mercogliano, Mercedes Tkach, Leandro Venturutti, Patricia Virginia Elizalde, Roxana Schillaci. Immunotherapy against breast cancer based on Stat3 blockade. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1346. doi:10.1158/1538-7445.AM2015-1346

Leptin-STAT3-G9a Signaling Promotes Obesity-Mediated Breast Cancer Progression.

Obesity has been linked to breast cancer progression but the underlying mechanisms remain obscure. Here we report how leptin, an obesity-associated adipokine, regulates a transcriptional pathway to silence a genetic program of epithelial homeostasis in breast cancer stem-like cells (CSC) that promotes malignant progression. Using genome-wide ChIP-seq and RNA expression profiling, we defined a role for activated STAT3 and G9a histone methyltransferase in epigenetic silencing of miR-200c, which promotes the formation of breast CSCs defined by elevated cell surface levels of the leptin receptor (OBR(hi)). Inhibiting the STAT3/G9a pathway restored expression of miR-200c, which in turn reversed the CSC phenotype to a more differentiated epithelial phenotype. In a rat model of breast cancer driven by diet-induced obesity, STAT3 blockade suppressed the CSC-like OBR(hi) population and abrogated tumor progression. Together, our results show how targeting STAT3-G9a signaling regulates CSC plasticity during obesity-related breast cancer progression, suggesting a novel therapeutic paradigm to suppress CSC pools and limit breast malignancy.

216. TLR9-Targeted STAT3 Silencing Abrogates Immunosuppressive Activity of Myeloid-Derived Suppressor Cells from Prostate Cancer Patients

Recent advances in immunotherapy of advanced human cancers underscored the need to eliminate tumor-induced immunosuppression. The myeloid-derived suppressor cells (MDSCs) are important negative regulators of T cell responses in advanced solid tumors, such as prostate cancers. Targeting MDSCs proved challenging due to heterogeneity and phenotypic similarities between myeloid cell lineages. Here, we identify a population of Lin–CD15HICD33LO granulocytic MDSCs that accumulate in patients’ blood during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8+ T cell proliferation, IFNg and Granzyme-B production. The circulating MDSCs have high levels of activated STAT3, a central immune checkpoint regulator. As shown by immunofluorescent microscopy, the CD15+/pSTAT3+ cells are also important component of immune infiltrate in primary tumor and tumor-draining lymph node tissues in these patients. We previously generated an original strategy to silence genes specifically in TLR9+ cells using naked CpG-siRNA conjugates. Here, we demonstrate that the CpG-STAT3siRNA is quickly internalized by human MDSCs effectively reducing STAT3 expression. The TLR9-dependent STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on CD8+ T cells. Our further studies demonstrated that these effects resulted from reduced expression and enzymatic activity of Arginase-1, a downstream STAT3 target gene and T cell inhibitor. Overall, we demonstrate the feasibility of using targeted STAT3 blockade to alleviate MDSC-mediated immunosuppression without depletion of myeloid cells in prostate cancer patients. We anticipate that further development of this oligonucleotide strategy will generate safer and more effective immunotherapy for advanced prostate cancers and potentially other solid tumors.

CD4+ T cell STAT3 phosphorylation precedes acute GVHD, and subsequent Th17 tissue invasion correlates with GVHD severity and therapeutic response.

Th17 cells contribute to severe GVHD in murine bone marrow transplantation. Targeted deletion of the RORγt transcription factor or blockade of the JAK2-STAT3 axis suppresses IL-17 production and alloreactivity by Th17 cells. Here, we show that pSTAT3 Y705 is increased significantly in CD4(+) T cells among human recipients of allogeneic HCT before the onset of Grade II-IV acute GVHD. Examination of target-organ tissues at the time of GVHD diagnosis indicates that the amount of RORγt + Th17 cells is significantly higher in severe GVHD. Greater accumulation of tissue-resident Th17 cells also correlates with the use of MTX- compared with Rapa-based GVHD prophylaxis, as well as a poor therapeutic response to glucocorticoids. RORγt is optimally suppressed by concurrent neutralization of TORC1 with Rapa and inhibition of STAT3 activation with S3I-201, supporting that mTOR- and STAT3-dependent pathways converge upon RORγt gene expression. Rapa-resistant T cell proliferation can be totally inhibited by STAT3 blockade during initial allosensitization. We conclude that STAT3 signaling and resultant Th17 tissue accumulation are closely associated with acute GVHD onset, severity, and treatment outcome. Future studies are needed to validate the association of STAT3 activity in acute GVHD. Novel GVHD prevention strategies that incorporate dual STAT3 and mTOR inhibition merit investigation.

STAT3-mediated MMP-2 expression is required for 15-HETE-induced vascular adventitial fibroblast migration

ObjectiveVascular adventitial fibroblasts (VAFs) migration was involved in neointima formation, and increased 15-HETE levels contributed to vascular remodeling. However, how 15-HETE-induced VAF migration was not clear. Methods and results15-HETE-stimulated VAF phenotypic changes and migration as measured by the wound healing assay required STAT3 phosphorylation. JNK1 and CREB inhibition blocked 15-HETE-induced STAT3 activation and VAF changes. 15-HETE-induced MMP-2 expression and secretion were analyzed by Western blot and ELISA, respectively. MMP-2 knockdown blocked VAF migration and phenotypic alterations. JNK1, STAT3 and CREB blockade suppressed 15-HETE-induced MMP-2 expression in VAFs. MMP-2 promoter activity was assessed by chromatin immunoprecipitation using anti-STAT3 antibodies, which demonstrated that STAT3 was essential for 15-HETE-induced MMP-2 expression. Rats that suffered from hypoxia injury with or without treatment were examined. Pulmonary artery remodeling was obviously observed, and even the media was broken. MMP-2-positive staining was observed in the adventitia and intima. MMP-2 Serum secretion was enhanced as detected by ELISA, and MMP-2 and α-SMA protein expressions were increased after inducing hypoxia in the rats, which was restored in rats that had been administrated with NDGA. ConclusionThese results reveal that STAT3-mediated MMP-2 expression is required for 15-HETE induced-VAF migration.

Targeting Blockage of STAT3 in Hepatocellular Carcinoma Cells Augments NK Cell Functions via Reverse Hepatocellular Carcinoma–Induced Immune Suppression

STAT3 is an important transcriptional factor for cell growth, differentiation, and apoptosis. Although evidence suggests a positive role for STAT3 in cancer, the inhibitory effects of tumor STAT3 on natural killer (NK) cell functions in human hepatocellular carcinoma are unclear. In this study, we found that blocking STAT3 in hepatocellular carcinoma cells enhanced NK-cell antitumor function. In the case of STAT3-blocked hepatocellular carcinoma cells, NKG2D ligands were upregulated, which promoted recognition by NK cells. Importantly, the cytokine profile of hepatocellular carcinoma cells was altered; in particular, TGF-β and interleukin 10 (IL-10) expression was reduced, and type I interferon (IFN) was induced, thus facilitating NK-cell activation. Indeed, the cytotoxicity of NK cells treated with supernatant from STAT3-blocked hepatocellular carcinoma cells was augmented, with a concomitant elevation of molecules associated with NK cytolysis. Further experiments confirmed that the recovery of NK cells depended on the downregulation of TGF-β and upregulation of type I IFN derived from STAT3-blocked hepatocellular carcinoma cells. These findings demonstrated a pivotal role for STAT3 in hepatocellular carcinoma-mediated NK-cell dysfunction, and highlighted the importance of STAT3 blockade for hepatocellular carcinoma immunotherapy, which could restore NK-cell cytotoxicity in addition to its direct influence on tumor cells.

Could Signal Transducer and Activator of Transcription 3 be a Therapeutic Target in Obesity-Related Gastrointestinal Malignancy?

A large body of evidence has implicated the signal transducer and activator of transcription (STAT) family and particularly the ubiquitously expressed STAT3 protein in the pathogenesis of colorectal, hepatocellular, gastric and pancreatic carcinoma. Concomitantly, an increasing body of epidemiological evidence has linked obesity and its associated pro-inflammatory state with the development of gastrointestinal cancers. Visceral adipose tissue is no longer considered inert and is known to secrete a number of adipocytokines such as leptin, interleukin (IL)-6, IL-8, IL-1β and tumour necrosis factor-alpha (TNF-α) into the surrounding environment. Interestingly, these adipocytokines are strongly linked with the Janus kinase (JAK)/STAT pathway of signal transduction and there is experimental evidence linking IL-1β, IL-8 and TNF-α to JAK/STAT signaling in other tissues. The result is an up-regulation of a wide range of anti-apoptotic, pro-metastatic and pro-angiogenic genes and processes. This is particularly relevant for gastrointestinal malignancy as these factors have the potential to signal adjacent endothelial cells in a paracrine manner. This review examines the potential role of the STAT3 signaling pathway in the pathogenesis of obesity-related gastrointestinal malignancy and the potential therapeutic role of STAT3 blockade given its status as a signaling hub for a number of inflammatory adipocytokines.

STAT5 polarization promotes iTregs and suppresses human T-cell alloresponses while preserving CTL capacity

Alloreactivity negatively influences outcomes of organ transplantation or HCT from allogeneic donors. Standard pharmacologic immune suppression impairs T-cell function and jeopardizes the beneficial reconstitution of Tregs. Murine transplantation models have shown that STAT3 is highly expressed in alloreactive T cells and may be therapeutically targeted. The influence and effects of STAT3 neutralization in human alloreactivity, however, remain to be elucidated. In this study, S3I-201, a selective small-molecule inhibitor of STAT3, suppressed human DC-allosensitized T-cell proliferation and abrogated Th17 responses. STAT3 blockade significantly enhanced the expansion of potent iTregs and permitted CD8(+) cytolytic effector function. Mechanistically, S3I-201 polarized the ratio of STAT phosphorylation in favor of STAT5 over STAT3 and also achieved a significant degree of Foxp3 demethylation among the iTregs. Conversely, selective impairment of STAT5 phosphorylation with CAS 285986-31-4 markedly reduced iTregs. STAT3 represents a relevant target for achieving control over human alloresponses, where its suppression facilitates STAT5-mediated iTreg growth and function.

STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients

Myeloid-derived suppressor cells (MDSC) play a key immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). In this study, we characterized CD14+HLA-DR(-/lo) cells sorted from the tumors, draining lymph nodes, and peripheral blood of HNSCC patients. CD14+HLA-DR(-/lo) cells were phenotyped as CD11b+, CD33+, CD34+, arginase-I+, and ROS+. In all 3 compartments, they suppressed autologous, antigen-independent T cell proliferation in a differential manner. The abundance of MDSC correlated with stage, but did not correlate with previous treatment with radiation or subsites of HNSCC. Interestingly, MDSC from all 3 compartments showed high phosphorylated STAT3 levels that correlated with arginase-I expression levels and activity. Stattic, a STAT3-specific inhibitor, and STAT3-targeted siRNA abrogated MDSC’s suppressive function. Inhibition of STAT3 signaling also resulted in decreased arginase-I activity. Analysis of the human arginase-I promoter region showed multiple STAT3-binding elements, and ChIP demonstrated that phosphorylated STAT3 binds to multiple sites in the arginase-I promoter. Finally, rescue of arginase-I activity after STAT3 blockade restored MDSC’s suppressive function. Taken together, these results demonstrate that the suppressive function of arginase-I in both infiltrating and circulating MDSC is a downstream target of activated STAT3.

APE1/Ref-1 Regulates STAT3 Transcriptional Activity and APE1/Ref-1–STAT3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival

Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3–APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3–APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions.

Targeting Stat3 Abrogates EGFR Inhibitor Resistance in Cancer

EGF receptor (EGFR) is upregulated in most epithelial cancers where signaling through EGFR contributes to cancer cell proliferation and survival. The limited clinical efficacy of EGFR inhibitors suggests that identification of resistance mechanisms may identify new pathways for therapeutic targeting. STAT3 is upregulated in many cancers and activated via both EGFR-dependent and -independent pathways. In the present study, we tested the consequences of STAT3 inhibition in EGFR inhibitor-resistant head and neck squamous cell carcinoma (HNSCC) and bladder cancer models to determine whether STAT3 blockade can enhance responses to EGFR targeting. pSTAT3 expression was assessed in human HNSCC tumors that recurred following cetuximab treatment. Cetuximab-sensitive and -resistant cell lines were treated with a STAT3 decoy to determine EC(50) concentrations and the effects on STAT3 target gene expression by Western blotting. In vivo assays included evaluation of antitumor efficacy of STAT3 decoy in cetuximab-sensitive and -resistant models followed by immunoblotting for STAT3 target protein expression. Targeting STAT3 with a STAT3 decoy reduced cellular viability and the expression of STAT3 target genes in EGFR inhibitor resistance models. The addition of a STAT3 inhibitor to EGFR blocking strategies significantly enhanced antitumor effects in vivo. Biopsies from HNSCC tumors that recurred following cetuximab treatment showed increased STAT3 activation compared with pretreatment biopsies. These results suggest that STAT3 activation contributes to EGFR inhibitor resistance both in HNSCC and bladder cancer where concomitant targeting of STAT3 may represent an effective treatment strategy.

Selective Blockade of Pulmonary Epithelial Stat3 for the Treatment of Asthma

Asthma affects 22 million people in the US and 300 million worldwide. There is a clear need for alternative treatments, particularly for steroid-unresponsive disease. Respiratory epithelial Stat3 has previously been shown to contribute to asthma by stimulating both Th2 and Th17 responses. We performed studies to test the hypothesis that selective pulmonary epithelial Stat3 blockade can prevent asthma in a murine dust mite (DM) asthma model.

STAT3 Is Required for Notch-Induced Leukemogenesis, and Functions As a Critical Survival Effector in T-Cell Leukemia

Abstract 920The disruption of main signaling mediators of critical leukemia cell functions represents the most promising approach for the development of more effective targeted therapies for acute lymphoblastic leukemia (ALL). Increasing evidence suggests an important combinatorial role for cell autonomous, epigenetic and extrinsic factors in tumorigenesis and tumor progression. However, little is known on how oncogenic and microenvironmental signals are integrated within leukemia T-cells and how they influence T-cell leukemogenesis. Our studies in patients’ leukemia T-cells showed that oncogenic Notch enhances their fitness by regulating the transcription of IL-7Rα, thus increasing the sensitivity of leukemia cells to IL-7, which triggers survival and mitogenic pathways. Interestingly, analysis of the Notch1/CBF-1 complex regulating IL7-Rα transcription show that it contains STAT3, but not STAT5, the main STAT molecule implicated in lymphoid cell signaling. Further analyses showed that STAT3 signaling is active in patients’ T-ALL specimens and in leukemia cells from mice with Notch-induced leukemia. We observed that IL-7 signaling triggers STAT3 phosphorylation and transcriptional activity, regulating the expression of the STAT3 target genes Bcl-xL, survivin, Cyclin D3 and miR-21.We then performed studies using a genetic model of conditional STAT3 deletion in the hematopoietic system. We observed that STAT3 loss-of-function markedly decreased the response of T-lineage cells (including thymocytes) to IL-7 stimulation, even if these cells exhibit functional STAT5 signaling. Importantly, in comparison to wild-type animals, STAT3-null mice did not exhibit defects in common lymphoid progenitors (CLP) in the bone marrow (BM) or in T-cell development, namely on DN thymocyte subsets. Also, no changes were observed in STAT3-null mice in the surface expression or transcriptional levels of IL-7Rα in CLP or thymocytes. To investigate the putative role of STAT3 in leukemia development, we performed the Notch-induced tumor model using BM primitive hematopoietic cells from STAT3-null mice, which could be effectively transduced with the constitutively-active Notch (ICN) construct. We observed that STAT3 deletion significantly inhibited the development of T-cell leukemia, with animals surviving for long periods without evidence of leukemia. Importantly, this was not due to defects in homing or in early engraftment of transplanted cells, which was further documented using intravital microscopy.Finally, pre-clinical studies were performed to evaluate the therapeutic potential of STAT3 pharmacological targeting on leukemia T-cells. STAT3 blockade by the selective inhibitors STATTIC or S3I-201 or the JAK2 inhibitor WP-1066 abrogated STAT3 phosphorylation, inhibiting the transcription of Bcl-xL and Survivin. This inhibition resulted in marked inhibition of leukemia T-cells, which was observed in primary T-ALL cells, in a IL-7-dependent T-ALL line and in leukemia cells from mice carrying ICN-induced leukemia. Mechanistically, we observed that STAT3 blockade results in and massive leukemia cell apoptosis, which is associated with activation of the pro-apoptotic p53(Ser46), and PARP cleavage. Also, STAT3 silencing by shRNA prevented leukemia cell expansion. Strong anti-leukemia synergism was observed between low doses of STATTIC and the frontline therapeutic drug Dexamethasone, or selective PI3K or mTOR inhibitors.In summary, our studies indicate that STAT3 signaling plays an important role in the integration of IL-7 and Notch signals in T-cell leukemia, and unveiled an unexpected role for STAT3 in T-cell leukemogenesis. Finally, our work suggests that the targeted disruption of STAT3 represents a valid therapeutic approach in this cancer. Disclosures:No relevant conflicts of interest to declare.

Low-dose Erythropoietin reduces risk of heart failure induced by anti-cancer therapy

Heart disease can be caused by many different insults and injuries and often terminates in death from heart failure, an inability of the heart muscle to adequately supply blood to the body. A major aim in today's research is to define novel therapeutic strategies to limit myocardial damage and to enhance the ability of the heart muscle to regenerate and to heal itself. The anti-cancer drug doxorubicin (DOX) is used to treat a broad range of cancers but is limited in its use by severe side effects, most notably heart failure [1]. STAT3 is an important factor that promotes survival and migration of many tumor cell types and its pharmacological blockade hold good promise as a novel anti-cancer strategy [2, 3]. However, likewise to DOX, blocking STAT3 in the heart has been associated with heart failure in various experimental models [4, 5]. In our latest work, we found that low-doses of erythropoietin (EPO), a cytokine with multiple functions best known for its role to control the production of red blood cells, reduces the risk of heart failure mediated by DOX treatment or STAT3 blockade [6]. Our findings suggest that treatment with low-doses (hematocrit-inactive) synthetic EPO (CERA) during DOX treatment or STAT3 blockade is able to preserve the ability of cardiac progenitor cells to repair the vasculature of the heart and thereby to attenuate myocardial injury [6]. Recent studies have shown that the heart possesses intrinsic cardiac stem or progenitor cells that can contribute to regeneration and healing during disease and aging [7]. Little is known about the molecules and pathways that regulate stem cell proliferation and differentiation. In order to obtain a better understanding how the cardiac microenvironment impacts on the endogenous regeneration system of the adult heart, we studied phenotypic alterations of resident cardiac progenitor cells (identified by the expression of Sca-1) in heart failure as a consequence of DOX treatment or genetic ablation of STAT3 in cardiac muscle cells. We observed that in both heart failure models, cardiac progenitor cells displayed an impaired ability to form new blood vessels essential for oxygen delivery to the heart muscle[6]. Both models also exhibited reduced production of EPO in the heart muscle[6]. Interestingly, we found that cardiac progenitor cells bind EPO within the cardiac microenvironment, a feature that seems to be required to maintain the ability to differentiate into new blood vessels[6]. Importantly, administration of the synthetic EPO derivative CERA at a low-dosage (hematocrit-inactive) restored or preserved the endothelial differentiation potential of cardiac progenitor cells in mice with depleted STAT3 or in mice obtaining DOX treatment. Moreover, CERA preserved heart blood vessels and cardiac function in both mouse models[6]. Our data imply that short-term EPO administration at low-doses seems an attractive avenue to pursue for protecting the heart against chemotherapy-induced failure and might even have broader applications in cardiac regeneration[6]. However, further evaluation of EPO in tumor research is required, especially with regard to its effect on tumor growth and anti-tumor treatment strategies.