title: Synergistic interactions between HDAC and sirtuin inhibitors in human leukemia cells.
abstract: Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD(+)-independent HDACs are an established therapeutic target, the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited.
authors.affiliation: Department of Internal Medicine, University of Genoa, Genoa, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Cea
authors.forename: Debora
authors.initials: D
authors.lastname: Soncini
authors.forename: Floriana
authors.initials: F
authors.lastname: Fruscione
authors.forename: Lizzia
authors.initials: L
authors.lastname: Raffaghello
authors.forename: Anna
authors.initials: A
authors.lastname: Garuti
authors.forename: Laura
authors.initials: L
authors.lastname: Emionite
authors.forename: Eva
authors.initials: E
authors.lastname: Moran
authors.forename: Mirko
authors.initials: M
authors.lastname: Magnone
authors.forename: Gabriele
authors.initials: G
authors.lastname: Zoppoli
authors.forename: Daniele
authors.initials: D
authors.lastname: Reverberi
authors.forename: Irene
authors.initials: I
authors.lastname: Caffa
authors.forename: Annalisa
authors.initials: A
authors.lastname: Salis
authors.forename: Antonia
authors.initials: A
authors.lastname: Cagnetta
authors.forename: Micaela
authors.initials: M
authors.lastname: Bergamaschi
authors.forename: Salvatore
authors.initials: S
authors.lastname: Casciaro
authors.forename: Ivana
authors.initials: I
authors.lastname: Pierri
authors.forename: Gianluca
authors.initials: G
authors.lastname: Damonte
authors.forename: Filippo
authors.initials: F
authors.lastname: Ansaldi
authors.forename: Marco
authors.initials: M
authors.lastname: Gobbi
authors.forename: Vito
authors.initials: V
authors.lastname: Pistoia
authors.forename: Alberto
authors.initials: A
authors.lastname: Ballestrero
authors.forename: Franco
authors.initials: F
authors.lastname: Patrone
authors.forename: Santina
authors.initials: S
authors.lastname: Bruzzone
authors.forename: Alessio
authors.initials: A
authors.lastname: Nencioni
mesh_terms: D000178:Acrylamides / Q000494:pharmacology; D018952:Antigens, CD34 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D004357:Drug Synergism; D020868:Gene Silencing / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia / Q000201:enzymology* / Q000473:pathology*; D009243:NAD / Q000096:biosynthesis; D010880:Piperidines / Q000494:pharmacology; D037761:Sirtuins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000178:Acrylamides; D018952:Antigens, CD34; D056572:Histone Deacetylase Inhibitors; C480543:N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide; D010880:Piperidines; D051028:bcl-2-Associated X Protein; D009243:NAD; D037761:Sirtuins