Acute myeloid leukemia (AML) represents one of the most common adult leukemia and remains as a deadly disease for most patients. Inhibition of aberrant DNA methylation by decitabine or 5-azacitidine restores normal patterns of cell proliferation, differentiation and apoptosis, however, the clinical response is restricted to a minority of hematopoietic malignancies. Further, off-target uptake and low efficient delivery of chemotherapeutic agents leads to undesirable adverse effects. Hence, there is an immediate need for targeted delivery of novel therapeutic agents to leukemic cells. Our long-term goals are to develop innovative nanocarriers and 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 specific hypothesis is that there exists a synergistic effect among DNA hypomethylating agents (bortezomib, miR29b and Sp1 siRNA) when used as combination therapy and this synergism may be enhanced when delivered by liposome- or lipopolyplexe-based nanocarriers. We base that hypothesis on the observations that 1) miR29b disrupts all DNA methyltransferases (DNMTs) directly or indirectly in AML thereby leading to DNA hypomethylation. 2) proteasome inhibitor bortezomib depletes Sp1/NF:B-dependent DNMT1 transcription and abolishes DNMT3a and 3b expression via miR29b upregulation, in turn causing DNA hypomethylation. 3) Sp1/NF:B complex is a central regulator governing both miR29b and DNMT transcription. Inhibitors for both regulators display encouraging anti-DNMT activity. 4) targeted lipid-based nanocarrier delivery of chemo-compounds or oligo deoxyribonucleotides (ODNs) display more significant target downregulation thereby more pronounced anti-tumor activity in vitro and in vivo. Based on these observations, the experimental focus of this proposal is on the nanocarrier design, synthesis and optimization as well as the synergistic effect of bortezomib combined with miR29b or Sp1 siRNA.
The specific aims are designed to provide a comprehensive assessment of drug delivery efficacy and specificity in a variety of formulations and the evaluation of in vitro and in vivo pharmacological activity of bortezomib, miR29b and sp1 siRNA as monotherapy or combination therapy.
The specific aims are to: 1. Design, synthesize and optimize targeted liposomes (Lips) and lipopolyplexes (LPs) for delivering bortezomib and Sp1 siRNA or miR29b into AML cell lines and AML patient primary cells. We will develop suitable targeted liposomal formulations by i) synthesis of nanocarriers using polycarbonate membrane extrusion followed by remote-loading of the drug or newly developed microfluidic (MF) methods and then conjugated to targeting ligands, ii) characterization of targeted nanocarriers for the size and size distribution, drug delivery efficacy, cellular uptake and toxicity. 2. Determine the pharmacological activity of the combination of Lip-bortezomib with LPs-Sp1 siRNA or Lip-bortezomib with LPs-miR29b in AML cell lines and patient primary cells. We will demonstrate that simultaneously pharmacological modification of DNMT regulatory network by targeted-nanocarrier delivered DNA hypomethylating agents synergistically induces DNA hypomethylation in vitro using i) Western blot and quantitative PCR (qPCR), ii) LC/MS/MS and iii) MTS and PI/AV staining. 3. Perform preclinical in vivo evaluation of the pharmacological activity of the combination of Lip- bortezomib with LPs-Sp1 siRNA or Lip-bortezomib with LPs-miR29b in murine models. We will define the effective pharmacological dose of the combinations achieving synergisms in vivo by pharmacokinetic/pharmacodynamic (PK/PD) studies in leukemia-bearing mice using Western blot, qPCR, LC/MS/MS and the assessment of clinical efficacy. This project will be carried out through an interdisciplinary approach by investigators with expertise in translational research and experimental therapeutics (Liu, Marcucci and Garzon), PK/PD (Chan), nanoengineering (JLee and RLee). If successful, this investigation will advance the understanding of nanosciences, enhance the understanding of the roles of proteasome system, miR and DNA methylation in leukemogenesis and establish a fundamental concept for the inhibition of aberrant DNMT activities.
Acute myeloid leukemia (AML) is a malignant, heterogeneous disease characterized by proliferation with maturation arrest of myeloid blasts in bone marrow and blood. In the United States, the incidence of this disease is rising with the aging of general population, and previous studies demonstrated that epigenetic deregulation(abnormal DNA hypermethylation or histone deacetylation) plays a critically pathogenic role in AML. Inhibition of aberrant DNA methylation by decitabine or 5-azacitidine restores normal patterns of cell proliferation, differentiation and apoptosis, however, the clinical response is restricted to a minority of hematopoietic malignancies, and, unfortunately, the majority of patients continue to die from their disease. Further, off-target uptake and low efficient delivery of chemotherapeutic agents leads to undesirable adverse effects. Thus, there is an immediate need for targeted delivery of novel therapeutic agents to leukemia cells. This proposal, therefore, responds to this call by addressing the leukemogeneic role of aberrant DNA methyltransferase (DNMT) and abnormally expressed microRNA in AML and the regulatory role of microRNA and bortezomib, a FDA approved standard chemotherapeutic agent, in DNMT gene transcription, as well as developing the optimized nanocarrier to specifically and efficiently deliver DNA hypomethylating agents to tumor cells or tissues. The hypothesis is that the combination of bortezomib with microRNA or bortezomib with siRNA would result in synergistic antileukemic activity and this synergism would be further enhanced by nanotechnology. The overall aim of this research application is therefore to develop efficient vehicles for delivering antileukemic agents and explore novel therapeutic strategies to target human malignancies with aberrant DNA methyltransferase activities. We plan to prepare biomarker-targeted liposomal nanocarriers loaded with bortezomib or siRNA or microRNA to treat leukemia cells in vitro. We will use animal models to validate the antitumor activity of bortezomib or siRNA or microRNA, in turn identify the correct dosage and formulation for future clinical use. It is anticipated 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.
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|Shen, Na; Yan, Fei; Pang, Jiuxia et al. (2017) Inactivation of Receptor Tyrosine Kinases Reverts Aberrant DNA Methylation in Acute Myeloid Leukemia. Clin Cancer Res 23:6254-6266|
|Yan, F; Shen, N; Pang, J et al. (2017) A regulatory circuit composed of DNA methyltransferases and receptor tyrosine kinases controls lung cancer cell aggressiveness. Oncogene 36:6919-6928|
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|Yan, Fei; Shen, Na; Pang, Jiuxia et al. (2015) The DNA Methyltransferase DNMT1 and Tyrosine-Protein Kinase KIT Cooperatively Promote Resistance to 5-Aza-2'-deoxycytidine (Decitabine) and Midostaurin (PKC412) in Lung Cancer Cells. J Biol Chem 290:18480-94|
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|Mishra, Anjali; Liu, Shujun; Sams, Gregory H et al. (2012) Aberrant overexpression of IL-15 initiates large granular lymphocyte leukemia through chromosomal instability and DNA hypermethylation. Cancer Cell 22:645-55|
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