Abstract 1111: Synthetic microRNA (miR)-181a nanoparticles (NP) target RAS and sensitize cells to daunorubicin (DNR) in acute myeloid leukemia (AML)
Authors: Huang Xiaomeng , Sebastian Schwind , Eisfeld Ann-Kathrin , Jin Yan , Yu Bo , Hickey Christopher , Pang Jiuxia , Ramasamy Santhanam , Chan Kenneth , Perrotti Danilo , Muthusamy Natarajan , Byrd John , Blum William , Bloomfield Clara , Liu Shujun , Garzon Ramiro , Lee Robert , Lee Ly , Marcucci Guido 
Published: August 2012

Most AML patients (pts) fail to achieve long-term survival with current treatments. New therapeutic options are needed. We recently showed that high miR-181a expression is associated with better outcome in AML pts with normal karyotype, but the mechanisms underlying this achievement are unknown. Aberrant activation of RAS due to mutations or overexpression is frequent in human malignancies, including AML. MiR-181a has been shown to target KRAS. In this study, we show that it also targets NRAS. With luciferase reporter constructs spanning the predicted binding site in NRAS we found a 25% decrease in luciferase activity after co-transfection with miR-181a vs scramble control (sc) in 293T cells. Reduced KRAS & NRAS levels were found in miR-181a vs sc transfected OCI-AML3 and KG1a at mRNA (1.9 & 1.4 and 3.2 & 2.9 fold, respectively) & protein levels (2.2 & 1.5 and 1.7 & 1.3 fold, respectively). Thus increasing miR-181a may improve outcome in AML pts by inhibiting RAS signaling. Free synthetic miRs are degraded in bio-fluid & have limited cellular uptake. We developed a novel non-viral delivery system for synthetic miRs that overcomes these limitations. Polyethylenimine was used to capture synthetic miRs & form a polyplex core. An outside lipid layer of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, linoleic acid & 1,2-dimyristoyl-sn-glycerol-3-phosphoethanolamine, methoxypolyethylene glycol protected the miRs from degradation & clearance & facilitated their uptake. Since AML cells overexpress the transferrin (Tf) receptor, we conjugated the NPs with Tf for targeted delivery. Mature miR-181a in OCI-AML3 & KG1a cells were measured by qPCR after treatment with TfNP encapsulating precursor miR-181a (pM181a) molecules (TfNP181a) or sc (TfNPsc) at 0.5 nM. At 24h mature miR-181a levels increased 50 fold in OCI-AML3 & 15 fold in KG1a cells, while the levels of unrelated miR-140 remained unchanged. We found that delivered pM181a downregulated levels of KRAS & NRAS at mRNA and protein levels (OCI-AML3: 2 & 3 fold and 4.4 & 11.3 fold; KG1a: 1.7 & 1.6 fold, and 5 & 2.7 fold, respectively) vs. TfNPsc treatment. To analyze downstream effects we measured MAPK & p-MAPK protein & observed a TfNP181a-mediated decrease of p-MAPK by 2.9 & 3.2 fold in OCI-AML3 & KG1a vs. TfNPsc treatment. Next we tested if TfNP181a sensitized AML cells to chemotherapy (DNR). After priming with 0.5 nM TfNP181a or TfNPsc (for 48h) OCI-AML3 cells were treated with 0.2 uM DNR (for 72h). MTS assays showed decreased cell viability for priming: 44.5% vs 82.5% (p&lt;0.01). We confirmed these findings in primary pt blasts 62.4% (TfNP181a) vs. 96.5% (TfNPsc) (p&lt;0.01). In conclusion we report an effective NP-based delivery system for miRs. TfNP181a treatment showed RAS downregulation, RAS signaling inhibition & increased sensitivity to DNR in AML cells. TfNP181a may be a new therapeutic in AML & other malignancies involving RAS.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1111. doi:1538-7445.AM2012-1111