Despite progress made in understanding the biological mechanisms of myeloid leukemogenesis, acute myeloid leukemia (AML) remains a deadly disease for most of the patients. It is well documented that silencing of tumor suppressor genes (TSGs) mediated by aberrant DNA methyltransferase (DNMT)-dependent DNA hypermethylation plays a critical pathogenic role in the development and progression of AML. Azanucleosides, i.e., decitabine, have been recently approved by FDA as hypomethylating agents and positive clinical outcome has been achieved for some patients. However, the clinical response is restricted to a minority of hematopoietic malignancies. Hence, further studies are urgently required to explore novel therapeutic strategies or agents to overcome the dismal outcome. Our long-term goals are to elucidate the regulatory mechanisms controlling DNA methylation thereby leukemogenesis as a prerequisite to the development of therapeutic protocols that can be used to attenuate the disease process. The objectives of this grant application are to explore novel DNA hypomethylation agents (DNMTi) with diverse structures through distinct mechanisms from decitabine. The specific hypothesis behind the proposed research is that bioactive food component Thymoquinone (TQ) may achieve higher efficacy of DNA hypomethylation through modulating methylation regulator Sp1/NFkB/miR network. That hypothesis is based on the following observations: 1) miR29b directly disrupts DNMT3a/3b and indirectly abolishes DNMT1 via impairment of its transactivator Sp1, thereby leading to DNA hypomethylation. 2) Sp1/NFkB complex suppresses miR29b expression and positively correlates to DNMT level in AML cell lines and patient samples, suggesting that DNA methylation is under control of a protein-miR network involving NFkB activity, Sp1/NFkB complex, DNMTs and miRs. 3) plant-derived drugs displayed efficiently anti-leukemic activities with huge therapeutic potentials, while the conventional medicine has lots of side effects, 4) TQ is a bioactive constituent and acts as anticancer agent, with minimal level of toxicity to normal cells, by significantly blocking NFkB signaling pathways. Based on these observations, the experimental focus of this proposal is on the hypomethylating effect of TQ in vitro and in vivo.
The specific aims are designed to provide a comprehensive understanding of the mechanism(s) of TQ action(s) and to optimize the dose and schedule of administration of TQ effective against leukemic disease.
The specific aims of this application are to: 1. Elucidate the mechanism of TQ antileukemic activity by the demonstration that TQ functions as DNA hypomethylation agent through Sp1/NFkB/miR29b network in AML cell lines and patient primary blasts. We will demonstrate that pharmacological modification of Sp1/NFkB/miR29b regulatory network by TQ induces DNA hypomethylation in vitro using i) Western blot and quantitative PCR (qPCR), ii) LC/MS/MS and iii) MTS and PI/AV staining. 2. Perform preclinical in vivo evaluation of the pharmacodynamic and pharmacokinetic activity of TQ in leukemic mice models. We will i) define the effective pharmacological dose and schedule of TQ administration that will modulate the misbalanced Sp1/NFkB/miR29b network thereby DNA hypomethylation in vivo using Western blot, qPCR, and LC/MS/MS, ii) determine the plasma and intracellular PK parameters of TQ and correlate these with PD and clinical efficacy endpoints, iii) determine the clinical efficacy measured by survival duration. This project will be carried out through an interdisciplinary approach by investigators with expertise in translational research (Liu) and PK/PD studies (Chan). If successful, the novel DNMTi can be applied to solid tumor or non-proliferating malignancies, this investigation may advance our understanding of epigenetic changes mediated by specific bioactive food component, the roles of Sp1/NFkB, miR and DNA methylation in leukemogenesis, the molecular pathways of TQ action and help us to adequately evaluate the specificity of individual nutrient.

Public Health Relevance

Acute myeloid leukemia (AML), one of the most common types of leukemia in the western hemisphere, is characterized by expansion of malignant, non-functional immature cells (blasts) in the bone marrow (BM). This results in bone marrow failure with loss of normal number and activity of blood cells. The management of this disease is complex with only approximately 40% of the treated patients achieving a long-term remission. Lack of significant progress in the treatment of this disease in adults calls attention to the need for further development of novel therapeutic strategies. The aberrant DNA methylation of genes, which are important for the normal function of bone marrow, make contribution to the development and maintenance of AML and has been shown to be a suitable therapeutic target. In fact, azanucleosides, i.e., decitabine, are compounds approved by US Food and Drug Administration (FDA) and currently used to decrease DNA methylation. However, the limitations of these compounds such as activity only on the distinct fractions of proliferating malignant cells and early onset of chemoresistance have emerged as concerns for the effective use of these agents. Thus, discovery and development of novel DNA methylation-targeting strategies is essential to overcome the limitation of azanucleosides in clinical trial. This proposal, therefore, responds to this call by addressing the leukemogeneic role of DNA methyltransferase, protein kinase and abnormally expressed microRNA in AML as well as developing novel therapeutic strategies to overcome the dismal clinical outcomes. Further, plant-derived drugs are relatively non-toxic, inexpensive and available in an ingestive form with anti- cancer potential, preclinical and clinical studies demonstrated that the anticancer properties of bioactive components may be attributed to its influence on epigenetic processes through binding to DNMT1 enzymatic center or/and disrupting DNMT transcription. The hypothesis is that bioactive food component thymoquinone may achieve higher efficacy of DNA hypomethylation through modulating methylation regulator protein-microRNA network. The overall aim of this research application is therefore to gain novel and distinct therapeutic approaches to target abnormal DNA methylation in AML. Hence, we plan to test the pharmacologic activity of thymoquinone to improve the effectiveness of targeting abnormal DNA methylation in AML using cancer cell lines, AML patient primary blasts and animal models. We anticipate that this proposed work will be translated into the clinic for therapeutic use in patients'with leukemia unresponsive to current hypermethylation inhibitor therapy and, perhaps, for other solid tumors and non-proliferating cancers at the end of this proposal's funding cycle.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
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Developmental Therapeutics Study Section (DT)
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Arya, Suresh
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University of Minnesota Twin Cities
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Shen, Na; Yan, Fei; Pang, Jiuxia et al. (2014) A nucleolin-DNMT1 regulatory axis in acute myeloid leukemogenesis. Oncotarget 5:5494-509
Yan, F; Shen, N; Pang, J et al. (2014) Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation. Cell Death Dis 5:e1413