Our results indicate that sirtuins and classical HDACs cooperate in GANT61 leukemia cells to prevent apoptosis. Combined inhibition of the two types of HDACs results in a synergistic antileukemic activity with potential to have clinical applications. Our data indicate that sirtuin and HDAC inhibitors cooperate to the killing of human leukemia cells. A two-pronged mechanism is shown to contribute to this form of synergy. On the one hand, HDAC inhibitors upregulate the pro-apoptotic Bcl2-family protein Bax. In turn, this condition predisposes leukemia cells to apoptotic cell demise when SIRT1 is inhibited. These findings are in line with previous studies which showed that SIRT1 prevents Baxmediated apoptosis by causing its cytoplasmic sequestration by Ku70, and that SIRT1 blockade results in initiation of the intrinsic apoptotic pathway in the presence of Bax overexpression. We confirmed Bax��s role in the synergy between sirtuin and HDAC inhibitors in leukemia cells by overexpressing it and by showing that increased Bax amounts indeed augment the efficacy of sirtuin inhibitors. Moreover, CHIR-99021 silencing Bax by stable RNA interference was found to reduce the activity of sirtuin inhibitors and of their combination with VA. However, it was of interest to observe that, despite efficient Bax silencing, the activity of sirtuin inhibitors, alone or coupled to VA, was not fully abolished. These findings suggest that Bax-independent mechanisms may also play a role in the antileukemic activity of these drugs. This is not surprising given how broadly sirtuin- and HDAC-mediated protein modifications are predicted to affect protein expression and activity, resulting in increased predisposition to apoptotic programs in malignant cells. Therefore, since Nampt inhibitors for clinical uses are already available and have shown to be well tolerated, these could in principle replace sirtuin inhibitors in combination protocols with HDAC inhibitors. Importantly, since the concentrations of FK866, VA, BU, and vorinostat used in our experiments are within the pharmacological range, these drug combinations are predicted to also show activity in patients. Audrito and colleagues have recently reported that SIRT1 inhibition with nicotinamide has cytotoxic activity on B-CLL cells, and that this effect requires the presence of wild type p53. Previous studies showed that SIRT1 deacetylates p53, thereby preventing its transcriptional activity. Thus, SIRT1 inhibition was proposed to upregulate several p53-dependent pro-apoptotic factors in B-CLL cells, thereby promoting apoptosis. In our case, functional p53 did not appear to be required for the synergy between sirtuin inhibitors and HDAC inhibitors, since this form of cooperation was also observed in primary B-CLL cells with 17p deletion. Moreover, Jurkat cells, which carry a mutant p53, were also highly susceptible to the combination of sirtuin and HDAC inhibitors. Nonetheless, it remains conceivable that, at least in some of the cases we studied, increased p53-mediated transcription via SIRT1 inhibition did contribute to the observed synergistic cytotoxicity. As a matter of fact, the sirtuin inhibitors used in this study are not specific for SIRT1 and can also inhibit other sirtuins, including SIRT2, SIRT3, and, possibly, SIRT6. The same applies to the Nampt inhibitor FK866. SIRT6 involvement in the synergy with HDAC inhibitors is unlikely since Jurkat cells where SIRT6 had been silenced by RNA-interference failed to exhibit increased susceptibility to HDAC inhibitors. We suggest that the potential of other sirtuins as targets for treating leukemias is further investigated.