Targeting Mir-155 via The NEDD8-Activating Enzyme Inhibitor MLN4924: A Novel Therapeutic Approach For Acute Myeloid Leukemia (AML)
Authors: Khalife Jihane C , Radomska Hanna S , Saultz Jennifer , Santhanam Ramasamy , Huang Xiaomeng , Wang Hongyan , Curfman John P. , Wu Yue-Zhong , Hoellerbauer Pia , Alachkar Houda , Dorrance Adrienne M , Caligiuri Michael A. , Garzon Ramiro , Mendler Jason H. , Marcucci Guido 
Published: October 2019

Abstract
microRNA-155 (miR-155) is a short non-coding RNA that is associated with aggressive cancers and known to promote leukemogenesis. Recently, we have reported that aberrant miR-155 upregulation independently identifies high-risk cytogenetically normal AML patients, suggesting that this miR may also serve as a novel therapeutic target in AML. We and others have shown that miR-155 is positively regulated by NF-kB, a transcription factor that is constitutively activated in leukemic blasts and contributes to their aberrant proliferation and survival. MLN4924 (Millennium Pharmaceuticals Inc) is a novel drug that blocks neddylation and subsequent degradation of the NFkB inhibitor, IkBa, thereby inhibiting translocation of NF-kB to the nucleus. MLN4924 has demonstrated promising activity in early clinical trials for AML. We postulated that downregulation of miR-155 via NF-kB inhibition is at least in part responsible for the antileukemic activity of MLN4924.

Methods
AML cell lines and primary blasts were treated with 100-1000nM MLN4924 for 3-72 hrs. Messenger RNA and protein levels were determined by quantitative RT-PCR and immunoblotting, respectively. NF-kB activity was measured by luciferase reporter assays. Binding of NF-kB to the miR-155 promoter was detected by electromobility shift assay and Chromatin Immunoprecipitation. Transfection of miR-155 was performed using the siPORT TM NeoFXTM method. Apoptosis was assessed by Annexin V staining. For in vivo studies, we used NOD/SCID/g mice engrafted with MV4-11 cells. Two weeks after transplantation, the engrafted mice received intraperitoneal treatments of 180 mg/kg of MLN4924 every other day for 21 days. Mice in the control group were treated similarly with the vehicle alone (20% 2-hydroxypropyl-betacyclodextrin).

Results
In AML cell lines and primary AML patient blasts 12hr treatment with MLN4924 resulted in a ∼50% decrease of miR-155 expression at 300nM in THP-1 and MV4-11 cells and at 500nM in AML blasts (p<0.01). This was concomitant with a ∼ 50% and 70% decrease in NF-kB activity and binding to miR-155 promoter, respectively (p<0.01). These results correlated with a significant upregulation of mRNA levels of the key miR-155 target gene, SHIP1 [6-fold (p<0.05), 9-fold (p<0.01), and 2-fold (p<0.05) in THP-1 cells, MV4-11 cells, and AML patient blasts, respectively]. SHIP1 protein levels were increased in all samples as well. SHIP1 is a tyrosine phosphatase that blocks PI3K-mediated membrane localization of AKT, which is often aberrantly activated in human cancers, including leukemia. Thus, we postulated that MLN4924-induced upregulation of SHIP1 via miR-155 downregulation would also result in PI3K/AKT pathway inhibition. As predicted, MLN4924 treatment of AML cell lines and primary blasts resulted in inhibition of the active AKT, as evidenced by a decline of phospho-AKTThr308 levels. Furthermore, the pharmacologic activity of MLN4924 was inhibited by forced expression of miR-155 in THP-1 cells and AML blasts, as shown by a partial loss of SHIP1 upregulation and caspase-3 activation, thus preventing MLN4924-triggered induction of apoptosis (p<0.01) and decrease in cell viability (p<0.05). In vivo, mature miR-155 levels in the peripheral blood of xenografted mice decreased by 50% after 24hrs and 80% after 48hrs (p<0.01) from the first dose of MLN4924. Moreover, 21 days from the start of MLN4924 treatment, the average white blood cell count was significantly lower in the MLN4924-treated group (5,333 cells/ul ± 1040) compared with the vehicle-treated group (36,166 cells/ul ± 10,598; p< 0.01). The average spleen weight was also dramatically reduced in the MLN4924-treated group (58.06 mg ±12.74) compared with the control group (305.66 mg ±51.1; p<0.01). Importantly, MLN4924 significantly prolonged the survival of leukemic mice; median survival was 45.5 vs. 31 days for MLN4924-treated vs. control groups (p<0.0001, n=10 per group), respectively.

Conclusions
We showed that MLN4924 treatment of AML cells in vitro and in vivo resulted in decreased miR-155 expression, reactivation of its target gene, SHIP1, and concomitant inhibition of PI3K/AKT pathway. Our data also support that miR-155 downregulation is a critical component of MLN4924’s antileukemic activity. Thus, our work provides novel insight into MLN4924’s mechanism of action and the rationale for combining this drug with emerging anti-microRNA compounds.

Disclosures:
No relevant conflicts of interest to declare.