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.
Highlights
Histone deacetylases (HDACs) regulate the acetylation status of histones and other intracellular substrates
Our results indicate that sirtuins and classical HDACs cooperate in leukemia cells to prevent apoptosis
Throughout these experiments, sirtuin inhibitors and HDAC inhibitors were found to have partial cytotoxic activity in leukemia cells when used as single agents (Figure 1, S1, S2, S3, and Tables S1, S2, S3, S4)
Summary
Histone deacetylases (HDACs) regulate the acetylation status of histones and other intracellular substrates. The recently discovered class III HDACs are sirtuins (SIRT1-7) [3]. HDAC inhibitors, some of which have been available for decades, show antileukemic activity in vitro and in animal models, and underwent clinical evaluations, mostly for acute myelogenous leukemia (AML) and myelodysplastic syndromes [5,6, 7,8,9]. Overall, these agents are very well tolerated, which makes them suited for treating elderly patients or patients with relevant co-morbidities.
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