Proteasome Inhibitor Combination with Cytarabine or Arsenic Trioxide Can Potentiate Apoptosis in AML Cells.

Proteasome inhibitors block degradation of the NF-κB regulator, IκB, resulting in inhibition of NF-κB nuclear localization. Proteasome inhibitors have been tested in the setting of refractory acute leukemia, with proteasome inhibition demonstrated within leukemic blasts. Arsenic trioxide (AsO₃) has also been shown to inhibit NF-κB in leukemia. Since NF-κB is constitutively activated in primitive AML cells and serves as a regulator of many genes which encode proliferative and survival responses, we have begun to explore the effects of AsO₃ and bortezomib (PS-341, Velcade^TM ), on AML cell lines and primary cells. The farnesyl transferase inhibitors (FTIs) also inhibit AML proliferation, and their effects in combination with bortezomib have also been explored. Because FTIs may inhibit signal transduction pathways independent of those affected by NF-κB inhibition, and because FTIs may indirectly inhibit NF-κB function via Akt inhibition, the effects of combining these agents with other NF-κB agents on AML cells in vitro have also been explored. Bortezomib, at concentrations of 4nM or greater, inhibited NF-κB in AML cell lines and primary cells as did AsO₃ at concentrations of 1 nM or greater. NF-kappa B was measured by ELISA for p65 NF-κB activity. The nonpeptidomimetic FTI, R115777 (J&J), did not inhibit NF-κB at concentrations up to 100 nM, concentrations which effectively inhibit farnesylation of target proteins, whereas the FTI, L-744832 (Merck), was able to inhibit NF-κB expression at 1 μM from 24 to 72 hours of exposure. In the HL60 cell line, inhibited by FTI and bortezomib independently, the combination did not appear to have additive or synergistic effects. Furthermore, the effects of combined exposure to FTI and bortezomib on expression of activated caspase 3 or activated PARP cleavage were no greater than with bortezomib alone. Likewise, combination effects on expression of phosphorylated AKT or ERK were not observed. In contrast, the combination of bortezomib and AsO₃ resulted in decreased phospho-ERK expression and increased expression of cleaved PARP, suggesting increased apoptosis. When cytarabine, 100 nM was combined with bortezomib at 1 to 4 nM, no effect on timing of administration was noted, and apoptosis was increased with the combination as evidenced by an increase in cleaved PARP expression. Greater inhibition of proliferation was seen with this combination than with individual agents as demonstrated in MTT assays with combination index calculations suggesting synergism. With this combination, co-culture with an endothelial monolayer did not prevent the increased apoptosis noted with combined cytarabine and bortezomib. These studies suggest that future studies combining proteasome inhibition with standard chemotherapeutic agents or with other inhibitors of NF-κB like Arsenic trioxide may have greater antileukemic activity by inducing apoptosis in AML cells in vivo as well as in vitro, without obvious limitations of other targeted agents and drugs that are extruded by multridrug resistance transporters.