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B Cell Lymphoma Immunotherapy Using TLR9-Targeted Oligonucleotide STAT3 Inhibitors.

ABSTRACT: Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expression of downstream target genes, such as MYC and BCL2L1, in human and mouse lymphoma cells. We further demonstrated that injections (i.v.) of CpG-STAT3dODN inhibited growth of human OCI-Ly3 lymphoma in immunodeficient mice. Moreover, systemic CpG-STAT3dODN administration induced complete regression of the syngeneic A20 lymphoma, resulting in long-term survival of immunocompetent mice. Both TLR9 stimulation and concurrent STAT3 inhibition were critical for immune-mediated therapeutic effects, since neither CpG7909 alone nor CpG7909 co-injected with unconjugated STAT3dODN extended mouse survival. The CpG-STAT3dODN induced expression of genes critical to antigen-processing/presentation and Th1 cell activation while suppressing survival signaling. These effects resulted in the generation of lymphoma cell-specific CD8/CD4-dependent T cell immunity protecting mice from tumor rechallenge. Our results suggest that CpG-STAT3dODN as a systemic/local monotherapy or in combination with PD1 blockade can provide an opportunity for treating patients with B cell NHL.

SUBMITTER: Zhao X

PROVIDER: S-EPMC5910676 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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B Cell Lymphoma Immunotherapy Using TLR9-Targeted Oligonucleotide STAT3 Inhibitors.

Zhao Xingli X Zhang Zhuoran Z Moreira Dayson D Su Yu-Lin YL Won Haejung H Adamus Tomasz T Dong Zhenyuan Z Liang Yong Y Yin Hongwei H HH Swiderski Piotr P Pillai Raju K RK Kwak Larry L Forman Stephen S Kortylewski Marcin M

Molecular therapy : the journal of the American Society of Gene Therapy 20180117 3

Growing evidence links the aggressiveness of non-Hodgkin's lymphoma, especially the activated B cell-like type diffuse large B cell lymphomas (ABC-DLBCLs) to Toll-like receptor 9 (TLR9)/MyD88 and STAT3 transcription factor signaling. Here, we describe a dual-function molecule consisting of a clinically relevant TLR9 agonist (CpG7909) and a STAT3 inhibitor in the form of a high-affinity decoy oligodeoxynucleotide (dODN). The CpG-STAT3dODN blocked STAT3 DNA binding and activity, thus reducing expr ...[more]

PMID: 29433938

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Project description:BackgroundAutoimmune disorders, including Systemic Lupus Erythematosus (SLE), are associated with increased incidence of hematological malignancies. The matricellular protein osteopontin (OPN) has been linked to SLE pathogenesis, as SLE patients show increased serum levels of OPN and often polymorphisms in its gene. Although widely studied for its pro-tumorigenic role in different solid tumours, the role of OPN in autoimmunity-driven lymphomagenesis has not been investigated yet.MethodsTo test the role of OPN in the SLE-associated lymphomagenesis, the SLE-like prone Faslpr/lpr mutation was transferred onto an OPN-deficient background. Spleen from Faslpr/lpr and OPN-/-Faslpr/lpr mice, as well as purified B cells, were analysed by histopathology, flow cytometry, Western Blot, immunohistochemistry, immunofluorescence and gene expression profile to define lymphoma characteristics and investigate the molecular mechanisms behind the observed phenotype. OPN cellular localization in primary splenic B cells and mouse and human DLBCL cell lines was assessed by confocal microscopy. Finally, gain of function experiments, by stable over-expression of the secreted (sOPN) and intracellular OPN (iOPN) in OPN-/-Faslpr/lpr -derived DLBCL cell lines, were performed for further validation experiments.ResultsDespite reduced autoimmunity signs, OPN-/-Faslpr/lpr mice developed splenic lymphomas with higher incidence than Faslpr/lpr counterparts. In situ and ex vivo analysis featured such tumours as activated type of diffuse large B cell lymphoma (ABC-DLBCL), expressing BCL2 and c-MYC, but not BCL6, with activated STAT3 signaling. OPN-/-Faslpr/lpr B lymphocytes showed an enhanced TLR9-MYD88 signaling pathway, either at baseline or after stimulation with CpG oligonucleotides, which mimic dsDNA circulating in autoimmune conditions. B cells from Faslpr/lpr mice were found to express the intracellular form of OPN. Accordingly, gene transfer-mediated re-expression of iOPN, but not of its secreted isoform, into ABC-DLBCL cell lines established from OPN-/-Faslpr/lpr mice, prevented CpG-mediated activation of STAT3, suggesting that the intracellular form of OPN may represent a brake to TLR9 signaling pathway activation.ConclusionThese data indicate that, in the setting of SLE-like syndrome in which double strand-DNA chronically circulates and activates TLRs, B cell intracellular OPN exerts a protective role in autoimmunity-driven DLBCL development, mainly acting as a brake in the TLR9-MYD88-STAT3 signaling pathway.

Project description:BACKGROUND: The transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-?B, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear. RESULTS: The mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-?B, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-?B-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death. CONCLUSIONS: The inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.

Project description:BackgroundThe Janus kinase (JAK) and signal transduction and activation of transcription (STAT) signaling pathway is an attractive target in multiple cancers. Activation of the JAK-STAT pathway is important in both tumorigenesis and activation of immune responses. In diffuse large B-cell lymphoma (DLBCL), the transcription factor STAT3 has been associated with aggressive disease phenotype and worse overall survival. While multiple therapies inhibit upstream signaling, there has been limited success in selectively targeting STAT3 in patients. Antisense oligonucleotides (ASOs) represent a compelling therapeutic approach to target difficult to drug proteins such as STAT3 through of mRNA targeting. We report the evaluation of a next generation STAT3 ASO (AZD9150) in a non-Hodgkin's lymphoma population, primarily consisting of patients with DLBCL.MethodsPatients with relapsed or treatment refractory lymphoma were enrolled in this expansion cohort. AZD9150 was administered at 2 mg/kg and the 3 mg/kg (MTD determined by escalation cohort) dose levels with initial loading doses in the first week on days 1, 3, and 5 followed by weekly dosing. Patients were eligible to remain on therapy until unacceptable toxicity or progression. Blood was collected pre- and post-treatment for analysis of peripheral immune cells.ResultsThirty patients were enrolled, 10 at 2 mg/kg and 20 at 3 mg/kg dose levels. Twenty-seven patients had DLBCL. AZD9150 was safe and well tolerated at both doses. Common drug-related adverse events included transaminitis, fatigue, and thrombocytopenia. The 3 mg/kg dose level is the recommended phase 2 dose. All responses were seen among DLBCL patients, including 2 complete responses with median duration of response 10.7 months and 2 partial responses. Peripheral blood cell analysis of three patients without a clinical response to therapy revealed a relative increase in proportion of macrophages, CD4+, and CD8+ T cells; this trend did not reach statistical significance.ConclusionsAZD9150 was well tolerated and demonstrated efficacy in a subset of heavily pretreated patients with DLBCL. Studies in combination with checkpoint immunotherapies are ongoing.Trial registrationRegistered at ClinicalTrials.gov: NCT01563302 . First submitted 2/13/2012.

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B Cell Lymphoma Immunotherapy Using TLR9-Targeted Oligonucleotide STAT3 Inhibitors.

Publications

B Cell Lymphoma Immunotherapy Using TLR9-Targeted Oligonucleotide STAT3 Inhibitors.

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