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.