Recently, we reported that sequential administration of the benzamide HDAC inhibitor MS-275 and the nucleoside analog fludarabine (F) resulted in synergistic induction of apoptosis in human leukemia cells (Maggio et al, Cancer Res. 64, 2590; 2004). Herein, we extended this concept the hydroxamic acid HDAC inhibitor NVP-LAQ824, and investigated involvement of reactive oxygen species (ROS) as well as activation of the NF-kB and JNK pathways in mechanisms of lethality. Pre-exposure (24 h) of U937 cells to low, non-toxic concentrations (40nM) of NVP-LAQ824 followed by addition of F induced a dramatic increase in cell death (≥75%). Apoptosis correlated with activation of caspases 3, -7, -8 and -9, and mitochondrial injury (loss of mitochondrial membrane potential and cytoplasmic release of cytochrome c, AIF and Smac/DIABLO). No changes were detected in the levels of membrane-associated DR4 or DR5 death receptors. NVP-LAQ824 induced an early peak of ROS (30’-3h), which persisted for 4-6 hours. This was accompanied by increased activation of NF-kB (8-16h), which returned to basal levels after 24h, but was unaccompanied by changes in phosphor-JNK. On the other hand, exposure to F alone did not modify either ROS or JNK activation, but triggered a marked activation of NF-kB. However, administration of F to cells pretreated with NVP-LAQ824 (24 h), in contrast to F administered alone, generated a second increase in ROS (30’-3h) and phosphorylation of JNK. Interestingly, addition of F to NVP-LAQ824-pretreated cells failed to trigger NF-kB activation. Moreover, interference with NVP-LAQ824-induced NF-kB activation by either pharmacological (Bay 11-7082, an IkBα phosphorylation inhibitor) or genetic means (U937 cells expressing either the IkB super-repressor or a stable RelA/p65 siRNA), promoted the pro-apoptotic actions of F and phosphorylation of JNK. Additional studies employing the free-radical scavenger NAC and U937/TAM67 cells (c-Jun dominant negative) provided data supporting a model wherein preexposure of U937 to LAQ824 induces ROS, which in turn trigger an NF-kB antiapoptotic response. However, these events, through a yet to be determined mechanism, attenuated NF-kB activation by subsequently administered F, thereby enhancing JNK activation and apoptosis. Collectively, these results are in agreement with recent evidence demonstrating that HDACI-mediated modulation of NF-kB activation status represents a critical determinant of lethal responses to these agents, and further suggest that this phenomenon represents a mechanistically critical event in regulating NVP-LAQ824/fludarabine interactions.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]