Multiple BH3 mimetics antagonize antiapoptotic MCL1 protein by inducing the endoplasmic reticulum stress response and up-regulating BH3-only protein NOXA

Abstract

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

FIGURE 1.

Sensitization of leukemia cells to vinblastine by ABT-737 or GX15-070. A, ML-1 cells were incubated with 2.2 μm vinblastine (VB) with or without 0.1 μm ABT-737 or 5 μm GX15-070 for 0–6 h. The cells were stained with Hoechst 33342 and scored for apoptosis (top panel). Lysates from cells incubated with or without GX-15–070 were also analyzed for protein expression as indicated (bottom panel). B, ML-1 cells were incubated with 0–5 μm GX-15–070 for 6 h with or without 2.2 μm vinblastine and analyzed for apoptosis (top panel). Lysates from cells incubated with or without 2.2 μm vinblastine or 5 μm GX15-070 were analyzed for protein expression (bottom panel). The caspase inhibitor z-VAD-fmk was also added where indicated. C, NB4 cells were incubated with 2.2 μm vinblastine with or without 0.1 μm ABT-737 or 5 μm GX15-070 for 0–6 h. The cells were stained with Hoechst 33342 and scored for apoptosis (left panel). NB4 cells were incubated with 0–1 μm ABT-737 for 6 h with or without 2.2 μm vinblastine and analyzed for apoptosis (right panel).

FIGURE 2.

Cytotoxicity of BH3 mimetics. NB4 cells were incubated with each of the BH3 mimetics at the indicated concentrations for 0–24 h and subsequently stained with Hoechst 33342. The morphology of cells incubated with ABT-737 and 2-methoxy-antimycin A₃ was photographed on a fluorescent microscope. The scale bar represents 10 μm. The cells were scored for chromatin condensation, except in the case of 2-methoxy-antimycin A₃, which was scored for diffused chromatin morphology. The results reflect the averages of three independent experiments at most time points. S.E. are presented where n = 3, and they exceed the size of the symbol.

FIGURE 3.

Effect of ABT-737 on the localization of BCL2 family members. A, NB4 cells were incubated with 0–0.4 μm ABT-737 for 6 h, fractioned into pellet and supernatant, and probed for the indicated proteins. PARP was analyzed in whole cell lysates (WCL). B, THP-1, U937, Jurkat, and K562 cells were incubated with 0–10 μm ABT-737 for 6 h, fractionated, and analyzed for the localization of BAD. C, untreated K562 cells expressing S peptide-tagged BCL2 were lysed and incubated with S protein-agarose for 0–16 h, separated into pulldown fraction (PD) and leftover supernatant (S), and probed for the indicated proteins. D, K562 cells expressing S peptide-tagged BCL2 were incubated with 0–10 μm ABT-737 prior to pulldown with S protein-agarose for 16 h. The pulldown fractions were probed for the indicated proteins. E, cells treated as in D were immunoprecipitated with anti-MCL1 and probed for associated BIM. F, K562 cells were incubated with the indicated BH3 mimetics for 6 h and then incubated with S protein-agarose to pulldown BCL2 bound proteins. The lysates were then probed for associated BIM and BAD. G, K562 cells were incubated with ABT-737 alone, gossypol alone, or both in combination for 6 h, then immunoprecipitated for MCL1, and probed for associated BIM and NOXA. To prevent caspase-mediated proteolysis induced by the drug combination, the cells were also incubated in z-VAD-fmk (far-right lane) to demonstrate that the decrease in BIM was not due to caspase activity.

FIGURE 4.

Effect of BH3 mimetics on expression and localization of BCL2 family members. NB4 cells were incubated with each of the indicated BH3 mimetics for 6 h, fractionated into pellet and supernatant, and probed for the indicated proteins. PARP was analyzed in whole cell lysates (WCL). Pellet and supernatant fractions from NB4 cells incubated with 0.1 μm ABT-737 for 6 h (lanes A) were added as controls to each panel. The Western blot of MCL1 is a shorter exposure than presented in Fig. 3; hence MCL1 is only marginally visible when cells were incubated with ABT-737.

FIGURE 5.

Effect of BH3 mimetics on proteasome activity. A, NB4 cells were incubated with bortezomib for 0–24 h, and lysates were probed for the indicated proteins. B, NB4 cells were incubated with bortezomib or each of the BH3 mimetics for 6 h and assayed for proteasome activity (top panel). The error bars represent S.E. of at least three independent experiments. NB4 cells were also incubated with a range of concentrations of bortezomib or GX15-070 and assayed for proteasome activity and induction of NOXA (bottom panel). C, NB4 cells were incubated with 100 nm bortezomib, 20 μm gossypol, or 20 μm S1 for 6 h. Whole cell lysates (WCL) were made from which MCL1 was immunoprecipitated. The immunoprecipitated fraction (IP) and the remaining nonimmunoprecipitated supernatant (S) were probed for the indicated proteins. The error bars represent S.E. of three independent experiments where the value exceeds the size of the symbol.

FIGURE 6.

Relative changes in mRNA and proteins levels in response to BH3 mimetics. A, NB4 cells were incubated with bortezomib or each of the BH3 mimetics for 6 h. Total mRNA was purified from the cells and analyzed for relative mRNA expression levels of NOXA and MCL1 via quantitative RT-PCR. The error bars represent S.E. of three independent experiments. B, cells were incubated with bortezomib (Bort) or each of the BH3 mimetics for 0–6 h. Cell lysates were probed for expression of ER stress response proteins. 2mAA, 2-methoxy-antimycin A₃.

FIGURE 7.

Functional evidence for the selectivity of BH3 mimetics. A, CLL cells isolated from patients were incubated with each of the BH3 mimetics for 6 h, and then the lysates were probed by Western blot analysis. B, NB4 cells were incubated with gossypol, S1, or 2-methoxy-antimycin A₃ (2mAA) with or without ABT-737 for 6 h prior to Western blot analysis.

FIGURE 8.

BAX/BAK dependence of BH3 mimetic-induced apoptosis. A, wild-type Jurkat and JurkatΔBAK cells were incubated with each of the BH3 mimetics for 0–72 h, stained with Hoechst 33342, and scored for chromatin condensation. The results reflect the average of three independent experiments at most time points. The S.E. values are presented where n = 3, and they exceed the size of the symbol. B, wild-type Jurkat and JurkatΔBAK cells were incubated with 0.3 μm ABT-737 plus gossypol, S1, or 2-methoxy-antimycin A₃ for 6 h, stained with Hoechst 33342, and scored for chromatin condensation. The cells were also incubated with ABT-737, gossypol, S1, or 2-methoxy-antimycin A₃ for 6 h and lysed for Western blot analysis.