The putative BH3 mimetic S1 sensitizes leukemia to ABT-737 by increasing reactive oxygen species, inducing endoplasmic reticulum stress, and upregulating the BH3-only protein NOXA

Abstract

S1 is a putative BH3 mimetic proposed to inhibit BCL2 and MCL1 based on cell-free assays. However, we previously demonstrated that it failed to inhibit BCL2 or induce apoptosis in chronic lymphocytic leukemia (CLL) cells, which are dependent on BCL2 for survival. In contrast, we show here that S1 rapidly increases reactive oxygen species, initiates endoplasmic reticulum stress, and upregulates the BH3-only protein NOXA. The BCL2 inhibitors, ABT-737, ABT-263, and ABT-199, have demonstrated pro-apoptotic efficacy in cell lines, while ABT-263 and ABT-199 have demonstrated efficacy in early clinical trials. Resistance to these inhibitors arises from the upregulation of anti-apoptotic factors, such as MCL1, BFL1, and BCLXL. This resistance can be induced by co-culturing CLL cells on a stromal cell line that mimics the microenvironment found in patients. Since NOXA can inhibit MCL1, BFL1, and BCLXL, we hypothesized that S1 may overcome resistance to ABT-737. Here we demonstrate that S1 induces NOXA-dependent sensitization to ABT-737 in a human promyelocytic leukemia cell line (NB4). Furthermore, S1 sensitized CLL cells to ABT-737 ex vivo, and overcame resistance to ABT-737 induced by co-culturing CLL cells with stroma.

Figure 1

S1 induces NOXA and sensitizes NB4 cells to ABT-737. a NB4 cells were incubated with 0–40 μM S1 for 6 h; NOXA expression was analyzed by western blotting. b NB4 cells were incubated with 0-100 nM ABT-737 with or without S1 for 6 h. PARP cleavage was used as a marker of apoptosis. c NB4 cells were transfected with 3 μg siRNA (control or NOXA) incubated for 48 h, and then incubated with S1, ABT-737 or both in combination for 6 h. PARP cleavage and NOXA were assessed by western blotting. d Cells from “c” were incubated as indicated for 6 h and scored for apoptosis. Survival is expressed as the percentage of cells which did not exhibit condensed chromatin.

Figure 2

S1 induces NOXA through ER stress signaling. a NB4 cells were incubated as indicated for 6 h. RNA was purified and subjected to PCR to assess XBP1 processing. b NB4 cells were incubated with S1 for 0-6 h. Protein levels were analyzed by western blotting. The mobility shift in PERK is indicative of phosphorylation and activation. c NB4 cells were transfected with control siRNA or siATF4 plus siATF3 and incubated for 48 h. Transfected cells were then incubated with S1 or bortezomib for 6 h and probed for the indicated proteins. d NB4 cells transfected as in “C” were incubated with ABT-737, S1, or both for 6 h and scored for apoptosis.

Figure 3

S1 increases reactive oxygen species, which is required for ER stress, NOXA induction, and sensitization to ABT-737. a NB4 cells were loaded with H₂DCFDA for 1 h, then incubated for 0-1 h with 0-20 μM S1 and analyzed by flow cytometry. Increases in mean FL-1 signal reflect increased ROS. Values represent mean and 1 SEM (n=3). b NB4 cells were incubated with H₂DCFDA, treated as indicated for 1 h, and scored for increases in ROS. A 2-h incubation with hydrogen peroxide (H₂O₂) was used as a positive control. The increased signal seen with GX15-070 is due to autoflourescence (see supplemental Fig S1). c NB4 cells were incubated with H₂DCFDA plus 0-10 mM NAC for 1 h. Cells were then incubated with S1 for 1 h and analyzed for changes in ROS. Error bars represent 1 SEM (n=3). d NB4 cells were incubated with 0-10 mM NAC for 1 h, and then incubated with S1 for 6 h. Proteins were probed as indicated. e NB4 cells were incubated for 1 h with 0-10 mM NAC and then incubated with ABT-737, S1, or both for 6 h. PARP cleavage was used as a marker for apoptosis.

Figure 4

S1 sensitizes CLL cells, but not normal lymphocytes, to ABT-737. a CLL cells were incubated with S1 and ABT-737 as indicated. Cells were then incubated with Hoechst 33342 and scored for condensed chromatin. Survival is calculated as the percentage of cells which did not exhibit condensed chromatin. Values represent the mean and 1 SEM (n=3). b Cells treated as in “a” were assessed for PARP cleavage. c CLL cells were isolated and co-cultured for 24 h on CD154⁺ stroma cells, and then treated as indicated and scored for apoptosis. Values represent the mean and 1 SEM (n=3). d Lymphocytes were isolated from healthy donors, treated as indicated, and scored for apoptosis. Values represent the mean and 1 SEM (n=2).