Therapeutic Targeting of the RAS-Pathway by Synthetic Mir-181a Nanoparticles in Acute Myeloid Leukemia (AML).

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 (TfNP 181a ) 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 TfNP 181a mediated inhibition of the RAS pathway we assessed the expression of total MEK & AKT and p-MEK & p-AKT. TfNP 181a 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 TfNP 181a vs TfNPsc was observed. TfNP 181a treatment also resulted in antileukemic activity. TfNP 181a 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 P miR-181a levels associated with improved treatment response we next tested whether TfNP 181a could sensitize AML cells to daunorubicin (DNR). After 24h priming with 10 nM TfNP 181a 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 TfNP 181a 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 TfNP 181a: 62.4% vs TfNPsc: 96.5% ( P in vivo tests of TfNP 181a are ongoing. In conclusion we first report that miR-181a targets KRAS and its downstream effector MAPK1 in AML. TfNP 181a treatment resulted in reduced RAS expression & signaling inhibition, antileukemic activity & increased sensitivity to chemotherapy in AML cells. Developing TfNP 181a 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.