Abstract 3558: The natural product silvestrol decreases oncogenic FLT3 and miR-155 levels in FLT3-ITD positive Acute Myeloid Leukemia (AML) cells
Authors: Alachkar Houda , Liu Shujun , Schwind Sebastian , Becker Heiko , Metzeler Klaus H. , Hickey Christopher , Pang Jiuxia , Whitman Susan P. , Chan Kenneth K. , Garzon Ramiro , Lucas David M. , Perrotti Danilo , Wu Lai-Chu , Grever Michael R. , Kinghorn A D. , Marcucci Guido 
Published: February 2011

Abstract
Despite improvement in our understanding of leukemogenic mechanisms, the majority of AML patients (pts) fail to achieve long-term survival and die of their disease. Therefore, new therapeutic options are needed. Silvestrol, a natural product isolated from Aglaia foveolata, has been shown to have potent antitumor activity, most likely through inhibition of initiation of translation. Promising synergistic effects of silvestrol with chemotherapy were shown in AML, but the exact mechanism mediating the antileukemic activity remains to be fully elucidated. Recently, microRNAs (miRs), short non-coding RNAs, have been recognized to contribute to leukemogenesis by inhibiting expression of targeted tumor suppressor genes. Therefore, we hypothesized that the antileukemic effect of silvestrol may at least be partly mediated by activity on miRs. We investigated the effects of silvestrol on the expression of a panel of miRs previously linked to AML (miR-29a, miR-29b, miR-29c, miR-181a, miR-34a & miR-155) by quantitative RT-PCR in representative AML cell lines (MV4-11, Kasumi-1, K562). Among the miRs measured, miR-155 was the only one found to be altered, with &gt;40% reduction from baseline following 24h silvestrol treatment (50nM). MiR-155 has a known oncogenic effect in leukemia and is up-regulated in FLT3-ITD (internal tandem duplication)-positive blasts. FLT3 encodes a tyrosine kinase membrane receptor, and the FLT3-ITD mutation is carried by ∼30% of all AML pts. FLT3-ITD supports abnormal myeloid blast proliferation and survival, and predicts dismal outcome. Silvestrol treatment of FLT3-ITD-positive MV4-11 cells resulted in a sustained 40% decrease of miR-155 expression observed as early as 6h post-treatment, as well as down-regulation (80%) of FLT3 mRNA and protein expression compared with vehicle-treated controls as measured by quantitative RT-PCR and immunoblotting respectively. We confirmed this finding by demonstrating a significant reduction of FLT3 surface expression by flow cytometry. These effects were associated with a significant antiproliferative and apoptotic activity of silvestrol on MV4-11 cells (IC50 &lt;10nM at 48h) as measured by MTS assays and annexin/PI staining. We conclude that silvestrol has a potent cytotoxic activity on mir-155 and FLT3-ITD expressing AML cells. The finding that FLT3-ITD expression was down-regulated not only at the protein level but also at the RNA level suggests that the effect of silvestrol on FLT3 mutants may be in part mediated by previously unreported mechanisms other than translational inhibition. Our data support further investigation to elucidate these novel aspects of the antileukemic activity of silvestrol in specific subsets of AML and the design of therapeutic studies testing this compound alone and/or in combination with other tyrosine kinase inhibitors in FLT3-ITD-driven AML.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3558.