(732f) A Novel Anionic Nanoparticle Delivery System for Microrna-29b Targets FLT3 and KIT Receptor Tyrosine Kinase Expression in Acute Myeloid Leukemia | AIChE

(732f) A Novel Anionic Nanoparticle Delivery System for Microrna-29b Targets FLT3 and KIT Receptor Tyrosine Kinase Expression in Acute Myeloid Leukemia

Authors 

Huang, X. - Presenter, The Ohio State University
Yu, B., The Ohio State University
Lee, L. J., The Ohio State University
Pang, J., The Ohio State University
Santhanam, R., The Ohio State University
Wu, Y. Z., The Ohio State University
Chan, K. K., The Ohio State University
Blum, W., The Ohio State University
Bloomfield, C. D., The Ohio State University
Perroti, D., The Ohio State University
Byrd, J. C., The Ohio State University
Muthusamy, N., The Ohio State University
Garzon, R., The Ohio State University


MicroRNAs, small non-coding RNA molecules, contribute to the pathogenesis of various human cancers, including acute myeloid leukemia (AML). One of these microRNAs - miR-29b - is frequently downregulated in AML,has antileukemic activity and was shown to target genes involved in leukemogenesis.1 Furthermore, higher pretreatment levels of miR-29b were associated with improved clinical response to treatment with the anti-leukemic agent decitabine.2 Recently we demonstrated that miR-29b also downregulates the expression of the receptor tyrosine kinases (RTKs) KIT and FLT3 in AML.3,4 The aberrant activation of the RTKs KIT and FLT3, by mutations or overexpressed is frequently found in AML and much effort has been undertaken to target these RTKs. In AML, due to development of early resistance, most inhibitors of RTKs have not led to substantial clinical benefit. However, instead of targeting the activity of RTKs, decreasing their expression may be a valuable therapeutic alternative. Thus the artificial increase of miR-29b levels in AML blasts to downregulate the RTKs KIT and FLT3 may be of high clinical benefit. Although some miR delivery methods have been developed for solid tumors, these methods have limitations that prevent their use in leukemia therapy. Cationic lipid/polymer formulations frequently used in solid tumors have the tendency to accumulate in lungs, kidney and liver and may cause a non-specific immune response, for which the consequences in AML have not been fully explored yet. We developed a transferrin-conjugated anionic lipid-based nanoparticle (NP) system to deliver synthesized miR-29b mimic molecules to leukemia cells. Low-molecular-weight polyethylenimine (PEI) was used to provide a positive charge, which can easily capture the negatively charged miR molecules achieving high entrapment efficiency of more than 90%. The lipid-based carrier was made of 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE), Linoleic acid and 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (DMG-PEG). We chose linoleic acid because of its low binding affinity to small RNAs, which implies easy dissociation of miRs from the lipopolyplex core.Moreover, the NPs were protected from reticuloendothelial system clearance by DMG-PEG, which allowed long in vivo circulation times. Since AML blasts overexpress the transferrin receptor, we conjugated the NPs with transferrin (Tf) using a post-insertion method to increase the uptake by targeted delivery. The average size and zeta potential of empty NP and miR-loaded NP were 129.6 ± 1.02nm (± Standard Deviation [SD]) and -9.82 ± 1.53mV (± SD) and 137.6 ± 0.96nm and 22.48 ± 1.44mV, respectively. After the Tf-conjugation of the miR-loaded NP the size increased to 147.3 ± 4.74nm and the zeta potential was 5.82 ±1.87mV. To evaluate the efficiency of NP uptake we encapsulated FAM-fluorescent labeled miR and treated two AML cell lines (i.e. Kasumi-1 and MV4-11). Tf-conjugated NP (Tf-NP) treated cells showed the highest uptake by confocal microscopy and flow-cytometry (mean fluorescence intensity (MFI): 21.0 and 22.9 for Kasumi-1 and MV4-11 respectively) compared to non-Tf-conjugated NPs (MFI: 8.3 and 13.0). Next, we evaluated the efficiency of miR-29b mimics delivery to AML cell lines and primary patient blasts with the Tf-NP system. After treatment with miR-29b loaded Tf-NPs (Tf-NP-miR-29b) we observed an increase of intracellular levels of miR-29b, 420 folds increase in Kasumi-1 cells, 220 folds increase in MV4-11 cells and an average 1000 folds increase in patient blasts compared to negative control-loaded Tf-NP (Tf-NP-nc) treatment after 24 hours. The expression levels of an unrelated miR, i.e. miR-140, remained unchanged. To assess whether the increased miR-29b levels would lead to a decrease of KIT and FLT3 expression we performed western blot analysis 48 hours after treatment. Tf-NP-miR-29b treatment led to 3.4 and 2 folds decrease of FLT3 and 1.5 and 1.4 folds decrease of KIT in Kasumi-1 and MV4-11 cells respectively and on average 1.5 folds decrease of FLT3 and 2.5 folds decrease of KIT in cells derived from three AML patients compared to Tf-NP-nc treated cells. The Tf-NP-miR-29b-mediated downregulation of these targets had antileukemic effects on AML cells. Tf-NP-miR-29b treatment reduced growth (per day) from 32.2% to 25.3% (P=0.01) in Kasumi-1 cells and from 53.0% to 43.9% (P=0.007) in MV4-11 cells, compared to Tf-NP-nc treatment. We also observed a reduction of numbers of colony formed by 55-66% after Tf-NP-miR-29b treatment after 2 weeks. The average numbers of colonies formed were 143±9(±SD) and 65±6 (P<0.001) for Kasumi-1 and 213±7 and 80±5 (P<0.001) for MV4-11 of Tf-NP-nc and Tf-NP-miR-29b treatment, respectively. Since we recently demonstrated that higher pretreatment levels of miR-29b were associated with improved clinical response to decitabine treatment,4 we also tested whether priming AML cells with Tf-NP-miR-29b prior to decitabine treatment would have additional therapeutic effect. Tf-NP-miR-29b treatment (for 48h) followed by 0.5 μM decitabine (for 48h) decreased the cell viability compared to Tf-NP-nc treatment or unprimed cells by 40% (P=0.001) and 30% (P=0.01), respectively in Kasumi-1 and by 16% (P=0.003) and 22% (P=0.001), respectively in MV4-11 cells. We confirmed these findings in three AML patient blasts, Tf-NP-miR-29b priming, followed by 5 μM decitabine treatment decreased cell viability for patient 1 by 20% (P=0.01), for patient 2 by 40% (P=0.003) and for patient 3 by 22% (P=0.002), respectively compared to Tf-NP-nc priming. In conclusion, we report here a novel anionic NP-based delivery system for miRs in AML. Our delivery system for miR-29b showed targeting activity against the important RTKs KIT and FLT3 that are associated with leukemogenesis, antileukemic effects and improved response to decitabine treatment in AML cells. The modulation of miR-29b levels with the here proposed delivery system as a single agent or in combination with other anti-cancer drugs - i.e. decitabine - may represent a promising new targeted treatment option for AML patients in the near future.

  1. Garzon R, Liu S, Fabbri M, et al. MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1. Blood. 2009;113(25):6411–6418.
  2. Blum W, Garzon R, Klisovic RB, et al. Clinical response and miR-29b predictive significance in older AML patients treated with a 10-day schedule of decitabine. PNAS. 2010;107(16):7473–7478.  
  3. Liu S, Wu L, Pang J, et al. Sp1/NFκB/HDAC/miR-29b Regulatory Network in KIT-Driven Myeloid Leukemia. Cancer Cell. 2010; 17(4): 333-347
  4. Blum W, Schwind S, Tarighat SS, et al. Clinical and Pharmacodynamic Activity of the Combination Bortezomib and Decitabine: a Phase I Trial in Patients with Acute Myeloid Leukemia. Blood. 2012; published ahead of print as doi:10.1182/blood-2012-03-413898.
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