In vitro activity: Cambinol is a potent SIRT (silent information regulator enzymes) inhibitor with IC50 values of 56 and 59 μM for SIRT1 and SIRT2, respectively. It inhibits NAD-dependent deacetylase activity of human SIRT1 and SIRT2. Consistent with the role of SIRT1 in promoting cell survival during stress, inhibition of SIRT1 activity with cambinol during genotoxic stress leads to hyperacetylation of key stress response proteins and promotes cell cycle arrest. Treatment of BCL6-expressing Burkitt lymphoma cells with cambinol as a single agent induced apoptosis, which was accompanied by hyperacetylation of BCL6 and p53. Because acetylation inactivates BCL6 and has the opposite effect on the function of p53 and other checkpoint pathways, the antitumor activity of cambinol in Burkitt lymphoma cells may be accomplished through a combined effect of BCL6 inactivation and checkpoint activation. Cambinol was well tolerated in mice and inhibited growth of Burkitt lymphoma xenografts. Inhibitors of NAD-dependent deacetylases may constitute novel anticancer agents..

Kinase Assay: The HDAC assay was done using bacterially expressed and purified glutathione S-transferase (GST) fusion proteins as previously described. The GST-SIRT2 expression plasmid was a gift of Dr. D. Moazed (Department of Cell Biology, Harvard Medical School, Boston, MA). SIRT1, SIRT3, and SIRT5 cDNA were generously provided by Dr. E. Verdin (Gladstone Institute of Virology and Immunology, San Francisco, CA). The genes were cloned into GST expression plasmids, expressed, and purified in bacteria. For deacetylation assays, chemically acetylated [3H]acetyl-H4 peptide was incubated with or without 500 μmol/L NAD+, 0.1 μg GST enzyme in the buffer containing 150 mmol/L NAD, 50 mmol/L Tris-HCl (pH 8.0), 1 mmol/L DTT, and 5% glycerol. After 3 hours of incubation at 37°C for SIRT1, SIRT2, SIRT3, and overnight incubation for SIRT5 (at 37°C), the reaction was quenched by the addition of 5 μL of 1 N HCl with 0.15 N acetic acid. Released [3H]acetic acid was extracted with 400 μL ethyl acetate and counted in 5 mL scintillation fluid. HDAC6 obtained from HeLa cells was transfected with HDAC6 expression plasmid by immunoprecipitation. HDAC6-specific rabbit polyclonal antibody and HDAC6 expression vector were a gift from Bob Eisenman (Fred Hutchinson Cancer Research Center).

Cell Assay: All cells were maintained at 37°C and 5% CO2; the appropriate medium was supplemented with 1% l-glutamine (Life Technologies, Carlsbad, CA) and 1% penicillin-streptomycin (Life Technologies). The nonadherent cell lines Dakiki, Daudi, Mutu1, Namalwa, Oku1, and Ramos were cultured in RPMI supplemented with 20% FCS. The EBV-transformed normal B cells B1, B2, and B3 and the cancer cells NCI H460 and Ovcar-3 were cultured in RPMI plus 10% FCS. HCT116, human foreskin fibroblasts, MCF-ADR, mouse embryonic fibroblasts (MEF) F61 and F80, 293/Phoenix, UACC 62, and Rat1a were maintained in DMEM plus 10% FCS. The chicken cells DT40 were grown in RPMI supplemented with 10% FCS, 1% chicken serum, and 10 μmol/L 2-mercaptoethanol. MCF-7 cells were maintained in DMEM with 10% FCS, 1% MEM nonessential amino acids (Life Technologies), 1% MEM sodium pyruvate (Life Technologies), 1 μg/mL human insulin (Life Technologies), 1 μg/mL hydrocortisone (Sigma), and 1 ng/mL human epidermal growth factor (Life Technologies) buffered with HEPES. Daudi, Dakiki, Namalwa, Ramos, HCT116, and NCI-H460 cells were obtained from American Type Culture Collection (Manassas, VA). The Burkitt lymphoma cells Oku1 and Mutu1 were received from Dr. I. Ruf (St. Jude Children's Research Hospital, Memphis, TN). The chicken cell lines DT40 cl18 and DT40 no. 77 were obtained from S. Takeda (CREST, Japan Science and Technology, Kyoto University, Kyoto, Japan). The normal B cell lines B1, B2, and B3 were EBV-transformed B cells obtained from Dr. Banker (Fred Hutchinson Cancer Research Center). The matched MEF F61 and F80 were received from Dr. Sanchez (Fred Hutchinson Cancer Research Center). Foxo3a MEF fibroblasts were generated from Foxo3a knockout mice