Diphtheria toxin fused to GM-CSF in combination with ARA-C exerts synergistic toxicity to human AML (HL60) cells

Human GM-CSF fused to a truncated diphtheria toxin (DT388-GM-CSF) showed a dramatic synergistic effect (combination index of 0.07) with Ara-C on HL60 human leukemia cells which was not seen with native GM-CSF. The enhanced cell kill occurred within 30 hr of exposure to both agents and did not involve detectable changes in Ara-C incorporation into cellular DNA. The synergistic toxic effect of Ara-C was not seen with 2 other protein synthesis inhibitors: ricin and cycloheximide. Enhanced cell kill with the DT388-GM-CSF/Ara-C combination was observed using both an 18 hr thymidine incorporation and a CFC assay to measure loss of proliferative potential and DAPI staining to detect nuclear fragmentation, but was not seen using the MTT assay to measure mitochondrial metabolism. Exposure to DT388-GM-CSF alone induced mitochondrial release and cytosolic accumulation of cytochrome c, and increased the PARP cleavage activity of caspase-3. Co-treatment with Ara-C enhanced both of these effects as well as the apoptosis induced by exposure to DT388-GM-CSF. However, the degree of enhancement of the effects on cytochrome c, caspase-3, and apoptosis of HL60 cells did not correlate with their reduced clonogenicity, Neither DT388-GM-CSF nor Ara-C treatment altered Bcl-2, Bcl-xl, Bax or FasR levels in HL60 cells, although DT388-GM-CSF increased Fas ligand expression by approximately 50%. Furthermore, enforced overexpression of Bcl-2 in the HL60 cells did not block the synergistic effect of DT388-GM-CSF and AraC. These findings suggest that ADP-ribosylation of elongation factor 2 by DT388-GM-CSF leads to a lowered apoptotic threshold to Ara-C and significantly enhances its activity against human AML cells. These observations also suggest clinical trials of DT388-GM-CSF in combination with Ara-C may be warranted in patients with AML.