Therapeutic Targeting of the RAS-Pathway by Synthetic Mir-181a Nanoparticles in Acute Myeloid Leukemia (AML).
Authors: Huang Xiaomeng , Schwind Sebastian , Eisfeld Ann-Kathrin , Yu Bo , Santhanam Ramasamy , Hoellerbauer Pia , Jin Yan , Hickey Christopher , Pang Jiuxia , Chan Kenneth K. , Perrotti Danilo , Muthusamy Natarajan , Byrd John C. , Blum William , Bloomfield Clara D. , Liu Shujun , Garzon Ramiro , Lee Robert J. , Lee L. James , Marcucci Guido 
Published: October 2019

Abstract
Abstract 2422
Today most AML patients (pts) still fail to achieve long-term survival. New therapeutic options are needed. We and others reported that high miR-181a expression associated with better outcomes in AML pts (Marcucci et al NEJM 2008; Schwind et al JCO 2010; Li et al Blood 2012), but the underlying mechanisms remain unclear. Aberrant RAS activation by mutations or overexpression is frequent in human malignancies, including AML. Previously KRAS was described as a direct miR-181a target in oral squamous cell carcinoma (Shin et al BBRC 2011). Here we confirmed KRAS as a direct miR-181a target & identified miR-181a-binding sites in the MAPK1 untranslated region (UTR) utilizing in silico tools & consequently identified MAPK1 as a new direct miR-181a target of the RAS signaling pathway. We observed respectively a 30±5% (±standard deviation; P=.003) & 35±2% (P=.0002) downregulation of luciferase activity after co-transfecting 293T cells with miR-181a mimic vs scramble control (sc) molecules and luciferase reporter constructs spanning the predicted miR-181a binding sites (seed sequences) in KRAS or MAPK1. Mutations in the seed sequences of KRAS or MAPK1 could rescue the miR-181a mimic induced downregulation. In the KG1a & OCI-AML3 AML cells with low endogenous miR-181a level but activated RAS-signaling, lentiviral miR-181a overexpression reduced KRAS protein by 2.8 & 2.1 fold and MAPK1 by 2.8 & 1.5 fold (normalized to β-actin & quantified by densitometry) respectively compared to sc expressing controls. Consistent with this result, lentiviral-based knock-down of miR-181a increased KRAS by 1.4 & 3.2 fold and MAPK1 protein by 2.3 & 1.8 fold in KG1a & OCI-AML3 cells respectively, compared to sc controls.
Since higher miR-181a associated with improved outcomes & miR-181a targets the RAS-pathway in AML, increasing miR-181a may have therapeutic value for AML pts. We formulated transferrin (Tf) targeted anionic lipid based lipopolyplex nanoparticles (NP) to deliver synthetic miR-181a mimic molecules to AML cells which overexpress Tf receptors, while overcoming RNA degradation & facilitating the cellular uptake of the miR molecules. Mature miR-181a in KG1a, OCI-AML3 cells & pts blasts (n=3) were measured by qPCR after treatment with Tf conjugated NP (TfNP) encapsulating miR-181a mimic molecules (TfNP181a) or sc (TfNPsc) at 10 nM. After 24h mature miR-181a levels increased 211±31, 880±10 & 145±19 fold in KG1a, OCI-AML3 & pts blasts (n=3), while levels of miR-181b & unrelated miR-140 remained unchanged. We found that delivered miR-181a downregulated KRAS & MAPK1 proteins (KG1a: 2.5 & 2.1 fold; OCI-AML3: 2.5 & 2.4 fold; pts blasts: 9.0 & 5.8 fold respectively) compared to TfNPsc treatment. To analyze downstream effects of TfNP181a mediated inhibition of the RAS pathway we assessed the expression of total MEK & AKT and p-MEK & p-AKT. TfNP181a compared to TfNPsc treatment decreased p-MEK by 3.2, 2.1 & 5 fold and p-AKT by 2.9, 3.6 & 6.5 fold in KG1a, OCI-AML3 & pts blasts. Furthermore, a 3.2, 4.7 & 3.1 fold reduction of the transcription factor MYC, which is regulated by MAPK in KG1a, OCI-AML3 & pts blasts treated with TfNP181a vs TfNPsc was observed.
TfNP181a treatment also resulted in antileukemic activity. TfNP181a compared to TfNPsc treatment led to reduced proliferation of KG1a (34±8% at day 4, P=.01) & OCI-AML3 (31±4% at day 4, P=.02) cells, and decreased colony formation (after 2 weeks) in both cell lines by 40±5% (P<.01) & 44±5% (P<.0001) respectively. Since higher miR-181a levels associated with improved treatment response we next tested whether TfNP181a could sensitize AML cells to daunorubicin (DNR). After 24h priming with 10 nM TfNP181a or TfNPsc, KG1a & OCI-AML3 cells were treated with 0.2 μM DNR for 72h. MTS assays showed decreased viability of the cells primed with TfNP181a vs TfNPsc: 66% vs 80.6% (P=.01) and 58% vs 81% (P=.02) for KG1a & OCI-AML3 respectively. We confirmed these findings in pts blasts primed with TfNP181a: 62.4% vs TfNPsc: 96.5% (P<.01). Preclinical in vivo tests of TfNP181a are ongoing. In conclusion we first report that miR-181a targets KRAS and its downstream effector MAPK1 in AML. TfNP181a treatment resulted in reduced RAS expression & signaling inhibition, antileukemic activity & increased sensitivity to chemotherapy in AML cells. Developing TfNP181a for miR-181a replacement therapy may represent a novel therapeutic approach to AML & may be extended to other malignancies involving RAS signaling.

Disclosures:
No relevant conflicts of interest to declare.