Submitted August 18, 2008
Accepted January 23, 2009
Caspase-8 dependent histone acetylation by a novel proteasome inhibitor, NPI-0052: a mechanism for synergy in leukemia cells
Claudia P. Miller, Sharmistha Rudra, Michael J. Keating, William G. Wierda, Michael Palladino, and Joya Chandra*
Department of Pediatrics Research, University of Texas M.D. Anderson Cancer Center, Houston, TX, United States
Department of Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, TX, United States
Nereus Pharmaceuticals, San Diego, CA, United States
* Corresponding author; email: jchandra{at}mdanderson.org.
Combination studies of histone deacetylase inhibitors (HDACi) and proteasome inhibitors are providing preclinical framework to build better strategies against hematological malignancies. Our previous work found that a novel proteasome inhibitor, NPI-0052, and HDACi synergistically induce apoptosis in leukemia cells in a caspase-8 and oxidant dependent manner. Here we extend those observations to primary leukemia cells and identify novel mechanisms of synergy. Since the proximal targets of NPI-0052 and HDACi are inhibition of proteasome activity and histone acetylation, we initially examined those biochemical events. Increased acetylation of histone-3 (H3) was detected in Jurkat and CLL primary cells treated with NPI-0052, alone or in combination with various HDACi (MS/SNDX-275 or vorinostat). Hyperacetylation by NPI-0052 occurred to a lesser extent in caspase-8 deficient cells and in cells treated with an antioxidant. These results indicate that NPI-0052 is eliciting caspase-8 and oxidative stress dependent epigenetic alterations. Additionally, real-time PCR revealed that MS/SNDX-275 repressed expression of the proteasomal 
5, 2 and 1 subunits, consequently inhibiting respective enzymatic activities. Overall, our results suggest that crosstalk by NPI-0052 and HDACi are contributing, along with caspase-8 activation and oxidative stress, to their synergistic cytotoxic effects in leukemia cells, reinforcing the potential clinical utility of combining these two agents.
