HDAC inhibitor-induced ROS generation mediates LAQ824/fludarabine lethality in human leukemia cells by enhancing DNA damage and inhibiting repair activity

A171 Previously, we reported that sequential administration of the HDAC inhibitor MS-275 (24h) followed by the nucleoside analog fludarabine (F) resulted in synergistic induction of apoptosis in human leukemia cells (Maggio et al, Cancer Res. 64, 2590; 2004). In the present study we extended this concept to a structurally different pan-HDAC inhibitor, the hydroxamic acid LAQ824, and investigated the role of reactive oxygen species (ROS) in this interaction. Pre-exposure (24 h) of U937 cells to non-toxic concentrations of LAQ (40nM) followed by addition of a marginally toxic concentration of F (0.4μM) induced a dramatic increase in cell death (≥75%), accompanied by activation of caspases 3, -7, -8 and -9, and mitochondrial injury. A time-course study of ROS generation revealed an LAQ824-induced early peak (30’-3h), which persisted for 4-6 hours; in contrast exposure to F alone or following LAQ824 pretreatment did not modify ROS levels. Analysis of proteins involved in ROS regulation revealed no changes in TRX, TRX-R or SOD1, while both Mn-SOD2 and the TRX binding protein VUDP1 were strongly induced by LAQ824. On the other hand, F alone or after LAQ824 did not modify expression of these proteins.. Notably, LAQ824/F-induced cell death was significantly diminished by the ROS scavengers NAC or the SOD2 mimic Mn-TBAP, indicating that LAQ824-mediated ROS generation played a functional role in lethality. Similar protection was observed in U937 cells ectopically expressing Mn-SOD2; conversely, cells expressing Mn-SOD2 in the antisense orientation displayed increased sensitivity to LAQ824 ± F. Recent studies have shown that HDACI-induced lethality may involve DNA damage but the basis for this phenomenon is not yet well established. To test whether LAQ824/F-induced ROS generation and subsequent lethality might be related to increased DNA damage, levels of γ-H2AX and p-ATM were monitored. Exposure of U937 cells to LAQ824 induced an early (4-8 h) increase in γ-H2AX which persisted over a period of 48 hours. While F alone modestly induced γ-H2AX after 24 h, preexposure to LAQ824 resulted in a dramatic, accelerated increase in DNA damage8-16 hours after F administration. Similar results were observed when p-ATM and ATM foci formation were monitored.. Notably, these effects were substantially reversed by coadministration of the ROS scavengers NAC or Mn-TBAP. In addition, LAQ824 induced a dramatic decrease in expression of the DNA repair proteins Ku86 and Rad50, and an increase in Ku70 acetylation.LAQ824/F-induced DNA damage was associated with the pronounced release of histone H1.2 from the nucleus to the cytosol, accompanied by subsequent Bak and caspase- 2 activation, suggesting that LAQ824-induced ROS generation may represent a critical event in HDACI-mediated DNA damage. Collectively, these findings support a model wherein exposure of LAQ824-pretreated cells to fludarabine results in a marked increase in basal levels of DNA damage as well as markedly impaired DNA repair activity. The resulting pronounced increase in DNA damage leads to the release of histone H1.2 into the cytosol, accompanied by activation of Bak and mitochondrial injury, culminating in apoptosis.