title: Silvestrol exhibits significant in vivo and in vitro antileukemic activities and inhibits FLT3 and miR-155 expressions in acute myeloid leukemia.
abstract: BACKGROUND
  Activating mutations [internal tandem duplication (ITD)] or overexpression of the FMS-like tyrosine kinase receptor-3 (FLT3) gene are associated with poor outcome in acute myeloid leukemia (AML) patients, underscoring the need for novel therapeutic approaches. The natural product silvestrol has potent antitumor activity in several malignancies, but its therapeutic impact on distinct molecular high-risk AML subsets remains to be fully investigated. We examined here the preclinical activity of silvestrol in FLT3-ITD and FLT3 wild-type (wt) AML.
  
  
  METHODS
  Silvestrol in vitro anti-leukemic activity was examined by colorimetric cell viability assay, colony-forming and flow cytometry assays assessing growth inhibition and apoptosis, respectively. Pharmacological activity of silvestrol on FLT3 mRNA translation, mRNA and protein expression was determined by RNA-immunoprecipitation, qRT-PCR and immunoblot analyses, respectively. Silvestrol in vivo efficacy was investigated using MV4-11 leukemia-engrafted mice.
  
  
  RESULTS
  Silvestrol shows antileukemia activity at nanomolar concentrations both in FLT3-wt overexpressing (THP-1) and FLT3-ITD (MV4-11) expressing AML cell lines (IC50 = 3.8 and 2.7 nM, respectively) and patients' primary blasts [IC50 = ~12 nM (FLT3-wt) and ~5 nM (FLT3-ITD)]. Silvestrol increased apoptosis (~4fold, P = 0.0001), and inhibited colony-formation (100%, P < 0.0001) in primary blasts. Silvestrol efficiently inhibited FLT3 translation reducing FLT3 protein expression by 80-90% and decreased miR-155 levels (~60%), a frequently co-regulated onco-miR in FLT3-ITD-positive AML. The median survival of silvestrol-treated vs vehicle-treated mice was 63 vs 29 days post-engraftment, respectively (P < 0.0001).
  
  
  CONCLUSIONS
  Silvestrol exhibits significant in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of FLT3 and miR-155 expression. These encouraging results warrant a rapid translation of silvestrol for clinical testing in AML.
authors.affiliation: Division of Hematology, Department of Medicine, The Ohio State University, Columbus, OH, USA. michael.grever@osumc.edu
authors.forename: Houda
authors.initials: H
authors.lastname: Alachkar
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Santhanam
authors.forename: Jason G
authors.initials: JG
authors.lastname: Harb
authors.forename: David M
authors.initials: DM
authors.lastname: Lucas
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Oaks
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hickey
authors.forename: Li
authors.initials: L
authors.lastname: Pan
authors.forename: A Douglas
authors.initials: AD
authors.lastname: Kinghorn
authors.forename: Michael A
authors.initials: MA
authors.lastname: Caligiuri
authors.forename: Danilo
authors.initials: D
authors.lastname: Perrotti
authors.forename: John C
authors.initials: JC
authors.lastname: Byrd
authors.forename: Ramiro
authors.initials: R
authors.lastname: Garzon
authors.forename: Michael R
authors.initials: MR
authors.lastname: Grever
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001752:Blast Crisis / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects*; D006801:Humans; D066298:In Vitro Techniques; D053631:Interleukin Receptor Common gamma Subunit / Q000502:physiology*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014315:Triterpenes / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D051941:fms-Like Tyrosine Kinase 3 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D001572:Benzofurans
chemical_list: D000970:Antineoplastic Agents; D053631:Interleukin Receptor Common gamma Subunit; D035683:MicroRNAs; D012333:RNA, Messenger; D014315:Triterpenes; D051941:fms-Like Tyrosine Kinase 3; C507942:Il2rg protein, mouse; C524942:MIRN155 microRNA, human; C489897:silvestrol; D001572:Benzofurans; C497970:FLT3 protein, human