Bcl-2 is a better ABT-737 target than Bcl-xL or Bcl-w and only Noxa overcomes resistance mediated by Mcl-1, Bfl-1, or Bcl-B

Abstract

The novel anticancer drug ABT-737 is a Bcl-2 Homology 3 (BH3)-mimetic that induces apoptosis by inhibiting pro-survival Bcl-2 proteins. ABT-737 binds with equal affinity to Bcl-2, Bcl-xL and Bcl-w in vitro and is expected to overrule apoptosis resistance mediated by these Bcl-2 proteins in equal measure. We have profiled ABT-737 specificity for all six pro-survival Bcl-2 proteins, in p53 wild-type or p53-mutant human T-leukemic cells. Bcl-B was untargeted, like Bfl-1 and Mcl-1, in accord with their low affinity for ABT-737 in vitro. However, Bcl-2 proved a better ABT-737 target than Bcl-xL and Bcl-w. This was reflected in differential apoptosis-sensitivity to ABT-737 alone, or combined with etoposide. ABT-737 was not equally effective in displacing BH3-only proteins or Bax from Bcl-2, as compared with Bcl-xL or Bcl-w, offering an explanation for the differential ABT-737 sensitivity of tumor cells overexpressing these proteins. Inducible expression demonstrated that BH3-only proteins Noxa, but not Bim, Puma or truncated Bid could overrule ABT-737 resistance conferred by Bcl-B, Bfl-1 or Mcl-1. These data identify Bcl-B, Bfl-1 and Mcl-1, but also Bcl-xL and Bcl-w as potential mediators of ABT-737 resistance and indicate that target proteins can be differentially sensitive to BH3-mimetics, depending on the pro-apoptotic Bcl-2 proteins they are complexed with.

Figure 1

Of all six pro-survival Bcl-2 proteins, Bcl-2 appears the optimal target for ABT-737. (a) MOLT-4 and J16 (Jurkat) T-ALL cell lines that had been transduced to stably express the indicated pro-survival Bcl-2 family members or empty control vector (EV) were treated with a dose range of ABT-737 (μM). Cell death was assessed by PI uptake, 48 h after ABT-737 addition. Gray dashed line indicates the curve fit for the EV dose–response data that is included in every graph for easy reference. Data shown are mean values+S.D. derived from one experiment with triplicate samples and are representative of two to three independent experiments. (b) EC₅₀ values of the dose–response curves in (a) as determined by curve fitting

Figure 2

Correction for protein turnover shows that Bcl-2 is a better target for ABT-737 than Bcl-xL and Bcl-w, while Bfl-1 and Mcl-1 are untargeted. (a) J16 cell line was transduced to stably express one of the indicated GFP-Bcl-2 fusion proteins or GFP control vector (EV) and cells were sorted on equal expression of GFP. Resulting stable cell lines with comparable expression of the GFP-tagged Bcl-2 proteins (Supplementary Figure 4) were subsequently treated with a dose range of ABT-737 (μM). Cell death was assessed by PI uptake, 48 h after ABT-737 addition. Data shown are mean values+S.D. derived from three independent experiments. (b) EC₅₀ values of the dose–response curves in (a) as determined by curve fitting

Figure 3

In J16 and MOLT-4 T-ALL, Bcl-2, Bcl-xL and Bcl-w mediate etoposide resistance, which can be alleviated by ABT-737 only in case of Bcl-2. (a) Single-agent treatment. J16 and MOLT-4 cell lines expressing empty vector (EV), or overexpressing one of the indicated pro-survival Bcl-2 proteins, were treated with the indicated doses of etoposide (μg/ml). Cell death was measured as PI uptake, 48 h after addition of the drug. Data shown are mean values+S.D. derived from one experiment with triplicate samples that is representative of three experiments. (b) Combined modality treatment. Cells were treated with a dose range of etoposide (0, 0.3, 0.6, 1.25, or 2.5 μg/ml for J16 and 0, 0.031, 0.063, 0.125, or 0.25 μg/ml for MOLT-4) in the absence of presence of ABT-737. ABT-737 was used at a concentration of 0.6 μM for J16 and at 0.16 μM for MOLT-4. Cell death was measured as PI uptake, 48 h after addition of the drugs. Data shown are mean values+S.D. derived from one experiment with triplicate samples that is representative of two experiments. CI were calculated as described in Materials and Methods and indicate synergy when <0.9

Figure 4

ABT-737 displaces Bad and Bax with greater efficacy from Bcl-2 than from Bcl-xL or Bcl-w. (a–d) HEK 293T cells were transfected to express each of the indicated HA-tagged pro-survival Bcl-2 proteins together with Flag-tagged Bim (a and b), Bax (c), or Myc-tagged Bad (d). At 24 h after transfection, ABT-737 or solvent control were added to the indicated final concentrations and cells were cultured for another 8 h in the presence of z-VAD-fmk (25 μM). Next, cells were harvested, lysed and subjected to immunoprecipitation (IP) with antibody directed at the HA tag. Before IP, samples were taken for control of expression of HA-tagged pro-survival and pro-apoptotic Bcl-2 proteins in total cell lysates (TCL). Samples of TCL and anti (α)-HA IP were separated by SDS-PAGE and subjected to western blotting with fluorochrome-conjugated α-HA, α-Flag and α-Myc antibodies. Imaging and quantification was done using the Odyssey Imager and associated software. Bar diagrams represent the ratio between the amounts of the pro-apoptotic and the pro-survival Bcl-2 family protein in the IP. The signal in the IP sample from mock-treated cells (control) was set at 100% for each of the pro-survival Bcl-2 proteins. Results shown are the mean+S.D. of three independent experiments. Asterisks denote statistical significant differences as determined by Student′s t-test (*P<0.05, **P<0.01, ***P<0.001)

Figure 5

Only Noxa synergizes with ABT-737 in Bcl-B, Bfl-1 and Mcl-1 overexpressing cells. J16 clones with Dox-inducible expression of the indicated BH3-only proteins and overexpression of Bcl-B, Bfl-1 or Mcl-1, were tested for interaction of the BH3-only protein with ABT-737. (a) Cell death assay: Cells were treated with ABT-737 at 80, 400, 2000, or 10000 nℳ, in the absence or presence of Dox (Bim and Noxa, 1 μg/ml; Puma, 0.25 μg/ml; tBid-C, 0.03 μg/ml). Cell death was assessed by PI uptake after 48 h. Data shown are mean values+S.D. derived from three independent experiments. CI indicate synergy. (b) Clonogenic assay: Cells were sorted at one cell per well into round-bottom 96-well plates, with medium alone (control), or added ABT-737 (1 μM), Dox (2 μg/ml), or both. Cells were allowed to proliferate for 3 weeks, and wells with evident colonies were scored positive. Colony formation in medium alone was set at 100%. Data shown are mean values+S.D. derived from three independent experiments

Figure 6

Proteasome inhibitor bortezomib increases Noxa expression, and works synergistically with ABT-737. (a) J16 and MOLT-4 cells, stably overexpressing Bcl-B, Bfl-1 or Mcl-1 that are not targeted by ABT-737, were incubated with ABT-737 (0,16, 80, 400, 2000, nM) in the absence or presence of bortezomib (15 nM and 5 nM for J16 and MOLT-4, respectively). After 24 h, cell death induction was assessed by PI uptake. Data shown are mean values+S.D. derived from three independent experiments. (b) J16 and MOLT-4 cells were treated for indicated periods of time with bortezomib supplemented with pan-caspase inhibitor to block cell death. Lysates were prepared and analyzed by western blotting for Noxa protein induction, using a Noxa-specific antibody. The blot was re-probed with an Actin-specific antibody to confirm equal loading