Synthetic oligonucleotides (ODN) expressing repetitive TTAGGG motifs patterned after hexameric sequences present at high frequency in mammalian teleomeres down-regulate the inflammatory immune responses elicited by a broad range of TLR ligands and the adaptive immune cell responses induced by polyclonal activators and antigens. These suppressive ODN are useful in the treatment of diseases characterized by over-exuberant immune responses, including septic shock and autoimmunity. Results from my group show that systemically administered Sup ODN alter the host immune milieu and can be harnessed to reduce susceptibility to inflammation-induced cancer. The initial focus of this research examined the effect of Sup ODN in the DMBA/TPA model of skin carcinogenesis. We found that Sup ODN significantly reduce both the number of mice that develop DMBA/TPA dependent papillomas and the number of papillomas/animal in this murine model of inflammation-associated tumorigenesis. To confirm this finding, we initiated studies in a completely different model system of inflammation-promoted tumorigenesis. First, we demonstrated that Sup ODN were effective in preventing/treating the life-threatening pulmonary inflammation caused by silicosis (a disease that affects many miners in the US and abroad). Epidemiologic studies of such miners suggest that the inflammation induced by silica particles (as well as asbestos and coal dust) increases their susceptibility to lung cancer induced by exposure to cigarette smoke. This led us to develop a novel model to examine whether exposure to silica dust plus NNK (the major carcinogen present in cigarette smoke) increase the risk of lung cancer in mice. Our findings demonstrate this to be the case (providing the first murine model of silica-induced sensitivity to lung cancer). More importantly, we show that treating silicotic inflammation with Sup ODN not only reduces pulmonary inflammation but returns cancer susceptibility to background. To clarify the mechanism by which Sup ODN inhibit tumor development, various measures of inflammation were examined. In both models studied, leukocyte infiltration and the production of pro-inflammatory cytokines and chemokines were significantly reduced whereas control ODN had no significant effect. We are extending these studies to include other agents that cause pulmonary inflammation and disease. We are also using microarray technology to identify the genes and regulatory networks triggered by suppressive ODN. These microarray studies indicate that very large numbers of genes are rapidly down-regulated following the administration of suppressive ODN. Two mechanisms by which Sup ODN broadly reduce gene expression are the focus of ongoing evaluation: i) that Sup ODN target TTAGGG and/or CCCTAA motifs present in the regulatory regions of critical genes (such as MAPKs) and ii) that Sup ODN inhibit the activity of bZIP proteins (regulatory DNA binding proteins that share a basic leucine zipper domain). In the context of MAPK regulation, mRNA encoding MAPK1, MAPK3 and MAPK14 (which stimulate the ERK-2, ERK-3 and p38 dependent pathways, respectively), as well as the transcription factors they regulate (ATF2, CREB1, NFKB1), contain TTAGGG and/or CCCTAA motifs in their regulatory regions that could be targeted by Sup ODN via an anti-sense mechanism . Since MAPKs exert a stabilizing effect on mRNAs encoding multiple inflammatory genes, down-regulating their expression could have a broad effect on the innate immune response. bZIP proteins are transcription factors that broadly influence gene expression. Transcription binding site analysis shows that many genes down-regulated by Sup ODN contain regulatory domains recognized by bZIP proteins (e.g. CREB1, CEBPA, and FOS). Indeed, 79% of the components in the TLR9 signaling pathway regulated by bZIP proteins are significantly down-regulated by Sup ODN (p <.0001). We examined whether Sup ODN inhibited the binding of bZIP proteins to their target DNA sequences and found that Sup ODN selectively inhibited the binding of three different classes of bZIP protein (CREB, C/EBPa and MAFg) to their target DNAs. Information gathered on the targets and mechanism(s) of action of suppressive ODN will support studies designed to explore their therapeutic utility.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010853-07
Application #
8763253
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$361,773
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Bayik, Defne; Gursel, Ihsan; Klinman, Dennis M (2016) Structure, mechanism and therapeutic utility of immunosuppressive oligonucleotides. Pharmacol Res 105:216-25
Klinman, Dennis M; Sato, Takashi; Shimosato, Takeshi (2016) Use of nanoparticles to deliver immunomodulatory oligonucleotides. Wiley Interdiscip Rev Nanomed Nanobiotechnol 8:631-7
Zhao, Jing; Mou, Yongshan; Bernstock, Joshua D et al. (2015) Synthetic Oligodeoxynucleotides Containing Multiple Telemeric TTAGGG Motifs Suppress Inflammasome Activity in Macrophages Subjected to Oxygen and Glucose Deprivation and Reduce Ischemic Brain Injury in Stroke-Prone Spontaneously Hypertensive Rats. PLoS One 10:e0140772
Wandu, Wambui S; Tan, Cuiyan; Ogbeifun, Osato et al. (2015) Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response. PLoS One 10:e0128906
Klinman, Dennis M (2015) Therapeutic implications of orally delivered immunomodulatory oligonucleotides. Mol Ther 23:222-3
Bode, Christian; Kinjo, Takeshi; Alvord, W Gregory et al. (2014) Suppressive oligodeoxynucleotides reduce lung cancer susceptibility in mice with silicosis. Carcinogenesis 35:1078-83
Takahashi, Ryohei; Sato, Takashi; Klinman, Dennis M et al. (2013) Suppressive oligodeoxynucleotides synergistically enhance antiproliferative effects of anticancer drugs in A549 human lung cancer cells. Int J Oncol 42:429-36
Bode, Christian; Yang, Xiang-Ping; Kiu, Hiu et al. (2013) Suppressive oligodeoxynucleotides promote the development of Th17 cells. PLoS One 8:e67991
Kaminski, John J; Schattgen, Stefan A; Tzeng, Te-Chen et al. (2013) Synthetic oligodeoxynucleotides containing suppressive TTAGGG motifs inhibit AIM2 inflammasome activation. J Immunol 191:3876-83
Klaschik, Sven; Tross, Debra; Klinman, Dennis M (2009) Inductive and suppressive networks regulate TLR9-dependent gene expression in vivo. J Leukoc Biol 85:788-95

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