Maciej Kujawski

Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity

The immune system can act as an extrinsic suppressor of tumors. Therefore, tumor progression depends in part on mechanisms that downmodulate intrinsic immune surveillance. Identifying these inhibitory pathways may provide promising targets to enhance antitumor immunity. Here, we show that Stat3 is constitutively activated in diverse tumor-infiltrating immune cells, and ablating Stat3 in hematopoietic cells triggers an intrinsic immune-surveillance system that inhibits tumor growth and metastasis. We observed a markedly enhanced function of dendritic cells, T cells, natural killer (NK) cells and neutrophils in tumor-bearing mice with Stat3−/− hematopoietic cells, and showed that tumor regression requires immune cells. Targeting Stat3 with a small-molecule drug induces T cell– and NK cell–dependent growth inhibition of established tumors otherwise resistant to direct killing by the inhibitor. Our findings show that Stat3 signaling restrains natural tumor immune surveillance and that inhibiting hematopoietic Stat3 in tumor-bearing hosts elicits multicomponent therapeutic antitumor immunity.

Persistently Activated Stat3 Maintains Constitutive NF-κB Activity in Tumors

NF-kappaB (RelA) is constitutively active in many cancers, where it upregulates antiapoptotic and other oncogenic genes. While proinflammatory stimulus-induced NF-kappaB activation involves IKK-dependent nuclear translocation, mechanisms for maintaining constitutive NF-kappaB activity in tumors have not been elucidated. We show here that maintenance of NF-kappaB activity in tumors requires Stat3, which is also frequently constitutively activated in cancer. Stat3 prolongs NF-kappaB nuclear retention through acetyltransferase p300-mediated RelA acetylation, thereby interfering with NF-kappaB nuclear export. Stat3-mediated maintenance of NF-kappaB activity occurs in both cancer cells and tumor-associated hematopoietic cells. Both murine and human cancers display highly acetylated RelA, which is associated with Stat3 activity. This Stat3/NF-kappaB interaction is thus central to both the transformed and nontransformed elements in tumors.

Regulation of the IL-23 and IL-12 balance by Stat3 signaling in the tumor microenvironment.

Interactions between tumor and immune cells either enhance or inhibit cancer progression. We show here that Stat3 signaling within the tumor microenvironment induces a procarcinogenic cytokine, IL-23, while inhibiting a central anticarcinogenic cytokine, IL-12, thereby shifting the balance of tumor immunity toward carcinogenesis. Stat3 induces expression of IL-23, which is mainly produced by tumor-associated macrophages, via direct transcriptional activation of the IL-23/p19 gene. Furthermore, Stat3 inhibits NF-kappaB/c-Rel-dependent IL-12/p35 gene expression in tumor-associated dendritic cells. Tumor-associated regulatory T cells (Tregs) express IL-23 receptor, which activates Stat3 in this cell type, leading to upregulation of the Treg-specific transcription factor Foxp3 and the immunosuppressive cytokine IL-10. These results demonstrate that Stat3 promotes IL-23-mediated procarcinogenic immune responses while inhibiting IL-12-dependent antitumor immunity.

In vivo delivery of siRNA to immune cells by conjugation to a TLR9 agonist enhances antitumor immune responses

Efficient delivery of small interfering (si)RNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We show that siRNA synthetically linked to a CpG oligonucleotide agonist of toll-like receptor (TLR)9 targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. When a CpG-conjugated siRNA that targets the immune suppressor gene Stat3 is injected in mice either locally at the tumor site or intravenously, it enters tumor-associated dendritic cells, macrophages and B cells. Silencing of Stat3 leads to activation of tumor-associated immune cells and ultimately to potent antitumor immune responses. Our findings demonstrate the potential of TLR agonist–siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.Efficient delivery of siRNA to specific cell populations in vivo remains a formidable challenge to its successful therapeutic application. We describe a novel siRNA-based approach – synthetically linking siRNA to an oligonucleotide TLR9 agonist – that targets and silences genes in TLR9+ myeloid cells and B cells, both of which are key components of the tumor microenvironment. Because Stat3 in tumor-associated immune cells suppresses antitumor immune responses and hinders TLR9-induced immune stimulation, we tested CpG-Stat3siRNA conjugates for anti-tumor effects. When injected locally at the tumor site or systemically through an intravenous route, the CpG-Stat3siRNA conjugates access tumor-associated dendritic cells, macrophages and B cells, inhibit Stat3 expression, leading to activation of tumor-associated immune cells, and ultimately potent anti-tumor immune responses. Our findings demonstrate the potential of TLR agonist-siRNA conjugates for targeted gene silencing coupled with TLR stimulation and immune activation in the tumor microenvironment.

Targeting STAT3 in adoptively transferred T cells promotes their in vivo expansion and antitumor effects

Adoptive cell therapy with engineered T cells to improve natural immune response and antitumor functions has shown promise for treating cancer. However, the requirement for extensive ex vivo manipulation of T cells and the immunosuppressive effects of the tumor microenvironment limit this therapeutic modality. In the present study, we investigated the possibility to circumvent these limitations by engineering Stat3 -deficient CD8(+) T cells or by targeting Stat3 in the tumor microenvironment. We show that ablating Stat3in CD8(+) T cells prior to their transfer allows their efficient tumor infiltration and robust proliferation, resulting in increased tumor antigen-specific T-cell activity and tumor growth inhibition. For potential clinical translation, we combined adoptive T-cell therapy with a Food and Drug Administration-approved tyrosine kinase inhibitor, sunitinib, in renal cell carcinoma and melanoma tumor models. Sunitinib inhibited Stat3 in dendritic cells and T cells and reduced conversion of transferred FoxP3(-) T cells to tumor-associated regulatory T cells while increasing transferred CD8(+) T-cell infiltration and activation at the tumor site, leading to inhibition of primary tumor growth. These data show that adoptively transferred T cells can be expanded and activated in vivo either by engineering Stat3-silenced T cells or by targeting Stat3 systemically with small-molecule inhibitors.

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells

Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte-associated antigen 4 (CTLA4(apt)) allows gene silencing in exhausted CD8⁺ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8⁺ T cells in the tumor milieu; therefore, CTLA4(apt) fused to a STAT3-targeting siRNA (CTLA4(apt)-STAT3 siRNA) resulted in internalization into tumor-associated CD8⁺ T cells and silencing of STAT3, which activated tumor antigen-specific T cells in murine models. Both local and systemic administration of CTLA4(apt)-STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4(apt)-STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4(apt)-STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4(apt)-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis.

Antitumor Activity of Targeting Src Kinases in Endothelial and Myeloid Cell Compartments of the Tumor Microenvironment

Purpose: Several Src family kinase (SFK) inhibitors have entered clinical trials based on their direct effects against tumor cells. Here, we characterize the effects of targeting Src kinases on the tumor microenvironment and how these effects influence tumor growth. Experimental Design: Human cancer cells grown in cell culture or in mice were treated with dasatinib, a small-molecule inhibitor of SFKs. Tumor cell, endothelial cell, and myeloid cell compartments within the tumor microenvironment were analyzed. Primary human endothelial cells and freshly isolated CD11b+/CD11c− myeloid cells from mice were treated with dasatinib in cell culture. Cellular functions and signaling pathways affected by dasatinib were evaluated. Results: Dasatinib was not cytotoxic in cell culture against the human cancer cell lines investigated here. However, dasatinib administration in human tumor–bearing mice suppressed tumor growth associated with increased tumor cell apoptosis, decreased microvessel density, and reduced intratumoral CD11b+ myeloid cells. Dasatinib directly inhibited motility and other functions of endothelial and myeloid cells, accompanied by the inhibition of phosphorylation of SFKs and downstream signaling. Tumor-infiltrating myeloid cells were identified as the major source of matrix metalloproteinase (MMP)-9 in the tumor microenvironment. Dasatinib treatment reduced MMP-9 levels in the tumor microenvironment through the simultaneous inhibition of recruitment of MMP9+ myeloid cells and MMP-9 gene expression in tumor-infiltrating myeloid cells. Conclusions: These findings suggest that Src kinase inhibitors such as dasatinib possess a previously unrecognized anticancer mechanism of action by targeting both host-derived endothelial and myeloid cell compartments within the tumor microenvironment. Clin Cancer Res; 16(3); 924–35

DNA damage kinetics and apoptosis in ivermectin-treated Chinese hamster ovary cells.

A comet assay was used to analyze DNA damage kinetics in Chinese hamster ovary (CHO‐K1) cells induced by antiparasitic ivermectin (IVM) and the IVM‐containing technical formulation Ivomec® (IVO; 1% IVM). Cells were treated with 50 µg ml–1 IVM and IVO for 80 min, washed and re‐incubated in antiparasiticide‐free medium for 0–24 h until assayed using the single‐cell gel electrophoresis assay (SCGE). Cell viability remained unchanged up to 3 h of incubation. After 6 h of treatment, cell survival decreased up to 75% and 79% in IVM‐ and IVO‐treated cultures, respectively, remaining unchanged within 12–24 h after treatment. For both anthelmintics, biphasic behavior in DNA damage occurred during the incubation time. A time‐dependent increase of IVM‐ and IVO‐induced DNA damage was observed within 0 to 3 h after pulse treatment, revealed by a progressive decrease of undamaged cells and an increase in slightly damaged and damaged cells. Finally, a time‐dependent decrease in IVM‐ and IVO‐induced DNA damage was revealed by a progressive decrease of slightly damaged cells and the absence of damaged cells simultaneously with an increase in the frequency of undamaged cells during the final 18 h of incubation. Flow cytometry analysis revealed that both compounds are able to induce a marked increase in early and late apoptosis. Based on our observations, we could conclude that the decrease in DNA lesions is mostly related to IVM‐induced cytotoxicity rather than attributable to a repair process. Copyright

CEACAM1 regulates the IL-6 mediated fever response to LPS through the RP105 receptor in murine monocytes

Systemic inflammation and the fever response to pathogens are coordinately regulated by IL-6 and IL-1β. We previously showed that CEACAM1 regulates the LPS driven expression of IL-1β in murine neutrophils through its ITIM receptor. We now show that the prompt secretion of IL-6 in response to LPS is regulated by CEACAM1 expression on bone marrow monocytes. Ceacam1-/- mice over-produce IL-6 in response to an i.p. LPS challenge, resulting in prolonged surface temperature depression and overt diarrhea compared to their wild type counterparts. Intraperitoneal injection of a 64Cu-labeled LPS, PET imaging agent shows confined localization to the peritoneal cavity, and fluorescent labeled LPS is taken up by myeloid splenocytes and muscle endothelial cells. While bone marrow monocytes and their progenitors (CD11b+Ly6G-) express IL-6 in the early response (<2 hours) to LPS in vitro, these cells are not detected in the bone marrow after in vivo LPS treatment due to their rapid and complete mobilization to the periphery. Notably, tissue macrophages are not involved in the early IL-6 response to LPS. In contrast to human monocytes, TLR4 is not expressed on murine bone marrow monocytes. Instead, the alternative LPS receptor RP105 is expressed and recruits MD1, CD14, Src, VAV1 and β-actin in response to LPS to produce IL-6. CEACAM1 negatively regulates RP105 signaling in monocytes by recruitment of SHP-1, resulting in the sequestration of pVAV1 and β-actin from RP105. This novel pathway and regulation of IL-6 producing by CEACAM1 defines a novel role for monocytes in the fever of mice to LPS. AUTHOR SUMMARY Fever is one of the most common signs of the immune response to pathogens. The fever response to LPS or endotoxin of gram-negative bacteria is mediated by the combined action of two cytokines, IL-1β and IL-6. Regulation of their production in response to LPS is an important area of investigation. While we previously showed that the regulation of IL-1β production in neutrophils is through the lymphocyte receptor CEACAM1, we were interested if a similar mechanism operated for IL-6. Using a mouse model in which the CEACAM1 gene was knocked out, we show that IL-6 is over-produced compared to normal mice, and that monocytes, rather than neutrophils were the principal IL-6 producing cells. Surprisingly, murine monocytes do not express TLR4, the most commonly studied receptor for LPS, but instead express the low affinity LPS receptor, RP105, a receptor common expressed on B-cells. Furthermore, we show that bone marrow monocytes are rapidly released into the blood and home to tissues throughout the body in response to LPS. These findings explain much of the confusion in the literature concerning the immediate source of IL-6 and the distinct differences between murine and human monocytes in their in responses to LPS.

Abstract 1637: STAT3 as a new molecular target for treating ABC-like DLBCL

Persistent STAT3 signaling contributes to malignant progression in many diverse human tumors. STAT3 has been found to be constitutively active in activated B cell (ABC)-like diffuse large B cell lymphomas (DLBCL). We investigated the effect of STAT3 silencing in human DLBCL Ly3 cells both in vitro and in vivo. For this purpose, we established stably transduced STAT3 shRNA-expressing lentivirus Ly3 cells and control lentivirus Ly3 cells. The stable expression of STAT3 shRNA resulted in 40-50% reduction of STAT3 protein levels compared to the control cells. In vitro experiments revealed that STAT3 downregulation was associated with a higher percentage of dead cells in STAT3 shRNA cells compared with the control cells, and this percentage was even higher in serum starvation conditions. STAT3 silencing resulted in inhibition of IL-10-inducible upregulation of c-Myc associated with downregulation of IL-10-dependent STAT3 activation and inhibition of IL-10-inducible cell growth. STAT3 inhibition also reduced adhesion to the bone marrow stroma layer and migration toward SDF-1 alpha. Moreover, analysis of inflammatory cytokines and receptors indicated a substantial downregulation of CXCL6 mRNA levels in STAT3 shRNA cells compared to the control cells. Tumors in control Ly3-bearing mice grew progressively, whereas tumors in STAT3 shRNA Ly3-bearing mice regressed 4-5 days after injection. This tumor regression was associated with Caspase-3-dependent apoptosis and significant reduction of STAT3 target genes at the protein level such as Mcl-1, c-Myc and Survivin. Moreover, immunofluorescence analysis of tumor specimens showed that while granulocytes and endothelial cells infiltrated into the control cells-bearing tumors, tumor bearing STAT3 shRNA cells showed less or no infiltration of these cells. We also observed reduction of IL-10 production and the secretion of other cytokines was altered as well. Thus, our results show that even partial down-regulation of STAT3 could achieve complete suppression of the tumorigenesis of ABC-like DLBCL Ly3 cells by affecting also the tumor microenvironment. Furthermore, we tested two STAT3 inhibitors, STATTIC and S31-201, in this system. The viability of Ly3 cells grown in vitro was reduced by both compounds tested in a dose-dependent manner and the antiproliferative effect was associated with inhibition of STAT3 phosphorylation. The antilymphoma activity of STATTIC was also observed in vivo. This is the first demonstration in vivo that direct STAT3 inhibition in ABC-like DLBCL suppresses tumor growth. While developing a small-molecule STAT3 inhibitor as well as siRNA delivery for clinical trials is still a challenge, our studies validate STAT3 as a good target for therapy in DLBCL and also establish that STAT3 siRNA-based gene therapy is a feasible approach for DLBCL. In Sum, our data support continued development of new STAT3 inhibitors as well as STAT3 siRNA delivery strategies for treatment of DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1637. doi:10.1158/1538-7445.AM2011-1637