(41f) Nicotinamide Increases the Megakaryocytic Maturation of Human Hematopoietic Stem Cells Primarily Due to Sirtuin Inhibition

Megakaryocytic cells (Mks), the precursors to platelets, are derived from hematopoietic stem cells (HSCs) and are among the least well-understood blood cell types. During differentiation, Mks increase in cytoplasmic volume and surface area, develop multi-lobated nuclei, become polyploid, form cytoplasmic extensions called proplatelets from which platelets are believed to arise, and undergo a complex apoptotic program. The process of becoming polyploid involves a variation of the mitotic cell cycle whereby the cells duplicate their DNA content without undergoing cytokinesis thus forming cells with 4N, 8N, 16N, etc. nuclei. Higher Mk DNA has been shown to be strongly correlated with greater platelet production. Thrombocytopenia, a deficiency in blood platelet levels, is associated with a variety of hematological malignancies and can also arise as a side effect of high-dose chemotherapy in cancer patients. In many cases, thrombocytopenia is associated with defective Mk maturation in terms of achieving high-ploidy Mk production in vivo. Elucidation of the factors that regulate Mk polyploidization will aid in developing treatments for Mk-related disorders. We have previously reported that nicotinamide, soluble vitamin B₃, increases the percentage of high-ploidy Mks ca. 3-fold and enhances proplatelet formation versus primary Mk cultures supplemented only with thrombopoietin. Nicotinamide is a well-established and potent inhibitor of sirtuin family of histone/protein deacetylases (SIRT) and has been found to be well tolerated by human subjects at high doses. The SIRT1/2 inhibitor cambinol increased Mk ploidy to a similar extent as nicotinamide without affecting Mk commitment or apoptosis. EX-527 and AGK2, selective inhibitors of SIRT1 and SIRT2, respectively, were approximately 30% as effective as nicotinamide at increasing Mk ploidy. This suggests that nicotinamide increases Mk ploidy via synergistic inhibition of SIRT1 and SIRT2. Nicotinamide also increases intracellular levels of NAD(H) 5-fold, but the application of an NAD⁺ de novo pathway precursor had minimal impact on ploidy, suggesting that the increase in intracellular NAD(H) is not the primary factor impacting Mk ploidy. We are currently investigating further downstream roles of the SIRT homologs in Mks such as acetylation of apoptosis related factors. Future plans include knockdown of SIRT1/2 in primary murine HSC cultures.