ABT-737 overcomes resistance to immunotoxin-mediated apoptosis and enhances the delivery of pseudomonas exotoxin-based proteins to the cell cytosol

Abstract

Pseudomonas exotoxin (PE)-based immunotoxins (antibody-toxin fusion proteins) have achieved frequent complete remissions in patients with hairy cell leukemia but far fewer objective responses in other cancers. To address possible mechanisms of resistance, we investigated immunotoxin activity in a model system using the colon cancer cell line, DLD1. Despite causing complete inhibition of protein synthesis, there was no evidence that an immunotoxin targeted to the transferrin receptor caused apoptosis in these cells. To address a possible protective role of prosurvival Bcl-2 proteins, the BH3-only mimetic, ABT-737, was tested alone or in combination with immunotoxins. Neither the immunotoxin nor ABT-737 alone activated caspase 3, whereas the combination exhibited substantial activation. In other epithelial cell lines, ABT-737 enhanced the cytotoxicity of PE-related immunotoxins by as much as 20-fold, but did not enhance diphtheria toxin or cycloheximide. Because PE translocates to the cytosol via the endoplasmic reticulum (ER) and the other toxins do not, ABT-737-mediated effects on the ER were investigated. ABT-737 treatment stimulated increased levels of ER stress response factor, ATF4. Because of its activity in the ER, ABT-737 might be particularly well suited for enhancing the activity of immunotoxins that translocate from the ER to the cell cytosol.

Fig 1

Response of DLD1 cells to immunotoxin treatment. The immunotoxin, HB21-PE40, was added at various concentrations as indicated, cells harvested and assayed as indicated below. A. Inhibition of protein synthesis was determined by measuring the incorporation of 3H-leucine into cells 24 hrs post treatment. Results are reported as CPM/well with error bars indicating one standard deviation. B. Cell viability using the WST-1 reagent was assayed after 48 hr. Shown are absorbance readings at 450 nm with error bars indicating one standard deviation. C. Cell energy levels, by measuring ATP, were determined after 48 hr. D. Apoptosis was assessed by measuring caspase 3/7 activity at 48 hr.

Fig 2

ABT-737 overcomes resistance of DLD1 cells. Immunotoxin or immunotoxin-ABT-737 combinations were added for 48 hr to DLD1 cells as indicated and assayed for viability, caspase activity or PARP cleavage. Cycloheximide (CHX) was added to inhibit protein synthesis. A. Cell viability using the WST-1 reagent shows enhanced killing with ABT-737. B. Caspase 3 activity in DLD1 cells treated with immunotoxin, immunotoxin plus ABT-737 or ABT-737 alone. C. Caspase 3 activity following CHX, CHX plus ABT-737 or ABT-737. D. Western blot of DLD1 lysates following treatments with immunotoxin alone, immunotoxin plus ABT or ABT alone. PVDF membranes were probed for PARP cleavage or the presence of Mcl-1. Equal protein amounts (~30 μgs) were loaded in each lane.

Fig 3

ABT-737 enhances immunotoxin activity in SKOV3 and KB3-1 cells. In panels A, B and C, cells were treated as indicated for 48 hr and viability was assessed using a WST-1 assay. D. KB3-1 cells were treated as indicated for 24 hr, cell lysates prepared and probed with antibodies to PARP, procaspase 3, Mcl-1 and tubulin.

Fig 4

ABT-737-mediated enhancement of KDEL-ending toxins. KB3-1 cells were incubated with various concentrations of (A) DT, (B) CHX or (C) an immunotoxin made with a truncated exotoxin from Vibrio cholerae and assayed for viability 48 hr post treatment. Toxin treatments were made alone or in combination with ABT-737 at the concentrations indicated. C. The SS1P immunotoxin was added to cells either alone or in combination with ABT-737 for 18 hr and then assayed for inhibition of protein synthesis.

Fig 5

ABT-737 causes ER stress. A. Lysates of ABT-treated DLD1 and KB3-1 cells were probed for the ER stress marker, ATF4. Lysates were prepared after 4 hr of treatment with either ABT-737 10 μM or 10 mM DTT.

Fig 6

ABT-737 and ABT-263 both exhibit immunotoxin-enhancing activity. A. ABT-737 enhances immunotoxin activity with even a short (4 hr) exposure to the combination. ABT-737 was added in combination with immunotoxins HB21-PE40 or SS1P for 4 hr, cells trypsinized and replated for 6 days. Cells that survived were visualized using methylene blue as the stain. B. ABT-263 enhances immunotoxin activity against DLD1 cells.