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. 2019 Jun;26(6):1037-1047.
doi: 10.1038/s41418-018-0183-7. Epub 2018 Sep 5.

BH3-only proteins are dispensable for apoptosis induced by pharmacological inhibition of both MCL-1 and BCL-XL

Georgia Greaves  1 , Mateus Milani  1 , Michael Butterworth  1 , Rachel J Carter  1 , Dominic P Byrne  2 , Patrick A Eyers  2 , Xu Luo  3 , Gerald M Cohen  1   4 , Shankar Varadarajan  5   6
Affiliations

BH3-only proteins are dispensable for apoptosis induced by pharmacological inhibition of both MCL-1 and BCL-XL

Georgia Greaves et al. Cell Death Differ. 2019 Jun.

Abstract

The impressive selectivity and efficacy of BH3 mimetics for treating cancer has largely been limited to BCL-2 dependent hematological malignancies. Most solid tumors depend on other anti-apoptotic proteins, including MCL-1, for survival. The recent description of S63845 as the first specific and potent MCL-1 inhibitor represents an important therapeutic advance, since MCL-1 is not targeted by the currently available BH3 mimetics, Navitoclax or Venetoclax, and is commonly associated with chemoresistance. In this study, we confirm a high binding affinity and selectivity of S63845 to induce apoptosis in MCL-1-dependent cancer cell lines. Furthermore, S63845 synergizes with other BH3 mimetics to induce apoptosis in cell lines derived from both hematological and solid tumors. Although the anti-apoptotic BCL-2 family members in these cell lines interact with a spectrum of pro-apoptotic BH3-only proteins to regulate apoptosis, these interactions alone do not explain the relative sensitivities of these cell lines to BH3 mimetic-induced apoptosis. These findings necessitated further investigation into the requirement of BH3-only proteins in BH3 mimetic-mediated apoptosis. Concurrent inhibition of BCL-XL and MCL-1 by BH3 mimetics in colorectal HCT116 cells induced apoptosis in a BAX- but not BAK-dependent manner. Remarkably this apoptosis was independent of all known BH3-only proteins. Although BH3-only proteins were required for apoptosis induced as a result of BCL-XL inhibition, this requirement was overcome when both BCL-XL and MCL-1 were inhibited, implicating distinct mechanisms by which different anti-apoptotic BCL-2 family members may regulate apoptosis in cancer.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
S63845 binds MCL-1 and induces apoptosis in several cancer cell lines. a S63845 exhibits a concentration-dependent shift in MCL-1 stabilization in a thermal stability assay. b S63845 and A-1210477 exhibit concentration-dependent apoptosis, as assessed by PS externalization using flow cytometry, in MCL-1-dependent cell lines, H929 and U-2946 after 4 h of exposure. c S63845 and A-1210477 fail to induce apoptosis in a BCL-XL-dependent cell line, K562, at 4 h, unlike A-1331852 (positive control). d MAVER-1, a BCL-2 dependent cell line, when exposed to the indicated BH3 mimetics for 4 h resulted in enhanced apoptosis. e Jurkat cells (wild type and deficient in the specified proteins), exposed to increasing concentrations of S63845 for 24 h exhibited caspase-9-dependent apoptosis. f Primary cells isolated from CLL patients, exposed to the indicated BH3 mimetics for 8 h, exhibited varying extents of apoptosis. g ABT-199 but not S63845 exhibits a concentration-dependent shift in BCL-2 stabilization in a thermal stability assay. Error bars = Mean ± SEM (standard error of the mean); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 2
Fig. 2
S63845 can synergize with ABT-199 to induce apoptosis in AML cell lines. a AML cell lines were exposed to a concentration range of S63845 with ABT-199 at a constant concentration of 100 nM for 24 h and assessed for PS externalization. Immunoprecipitation of MCL-1 and BCL-2 was carried out in b MV-4-11, c OCI-AML-3, and d HL-60 cells, following exposure to S63845 (30 nM for MV-4-11 and 100 nM for the other 2 cell lines) or ABT-199 (50 nM for MV-4-11 and 100 nM for the other 2 cell lines) for 6 h (MV-4-11) or 8 h (OCI-AML-3 and HL60). The eluted complexes were immunoblotted for the indicated proteins. The input represents the cell lysates and BC, beads control. Error bars = Mean ± SEM; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 3
Fig. 3
BH3-only proteins have different binding preferences in various solid tumor cell lines despite similar sensitivities to BH3 mimetics. a Solid tumor cell lines were treated with a concentration range of S63845 and a constant concentration of 100 nM of A-1331852 for 4 h and assessed for PS externalization. Immunoprecipitation of MCL-1 and BCL-XL was carried out in H1299 b, SUIT-2 c, and HCT116 d cells exposed to S63845 (100 nM) or A-1331852 (100 nM) for 2 h, and the eluted complexes were immunoblotted for the indicated proteins. * in the blots denotes non-specific bands. Error bars = Mean ± SEM (standard error of the mean); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 4
Fig. 4
BH3 mimetics induce apoptosis even in the absence of BH3-only proteins in HCT116 cells. a HCT116 cells, wild-type (WT) and deficient in 8 different pro-apoptotic BH3-only proteins (8KO), were exposed to A-1331852 (100 nM) in combination with increasing concentrations of S63845 for 4 h and assessed for PS externalization. b Same as (a) but the experiments were carried out with HCT116 WT cells or cells deficient in BAX, BAK or both (DKO). c Immunoprecipitation of MCL-1, BCL-XL and active-BAX in the indicated cells exposed to a combination of S63845 (100 nM) and A-1331852 (100 nM), following a 0.5 h pre-treatment with z-VAD.FMK (30 μM), were performed to assess BAX interaction. d The level of mitochondrial BAX in WT and 8KO cells following S63845 and A-1331852 treatment (both 100 nM) for 4 h was assessed by immunocytochemistry using an anti-BAX antibody. e Western blots of different molecular weight fractions from FPLC showing BAX oligomerization in HCT116 WT and 8KO cells upon exposure to S63845 (100 nM) and A-1331852 (100 nM) for 2 h. f Quantification and representative images of cytochrome c release in the indicated cell lines, from three independent experiments, following exposure to S63845 (100 nM) and A-1331852 (100 nM) for 4 h in the indicated cells. Error bars = Mean ± SEM (standard error of the mean); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 5
Fig. 5
Apoptosis regulated by BCL-XL requires BH3-only proteins. a Apoptosis was assessed by PS externalization in the indicated cell lines following exposure to A-1331852 (100 nM) and/or S63845 (100 nM) for the indicated times. HCT116 WT and 8KO cells were exposed to S63845 or A-1331852 (100 nM) for 24 h and assessed for b PS externalization, c cytochrome c release and d BAX translocation. For (c) and (d), cells were pre-treated with z-VAD.FMK (30 μM) for 0.5 h before exposure to BH3 mimetics. HCT116 WT and 8KO cells were e transfected with siRNA against BCL-XL and/or MCL-1 for 72 h and assessed for PS externalization or f exposed to A-1331852 (100 nM) and/or S63845 (100 nM) and the number of colonies formed after 7 d was counted and images taken using GelCount. Representative images from the clonogenic assay are shown in the right panel. Error bars = Mean ± SEM (standard error of the mean); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001
Fig. 6
Fig. 6
BCL-2 family members differ in their dependence on pro-apoptotic BH3-only members to exert their anti-apoptotic functions. In HCT116 cells, multiple BH3-only proteins, such as BIM, BID, BAD and PUMA are sequestered by BCL-XL, whereas NOXA is the only BH3-only protein bound to MCL-1. While the displacement of BH3-only proteins from BCL-XL following A-1331852 is sufficient to induce apoptosis in these cells, displacement of NOXA from MCL-1 following S63845 failed to induce apoptosis. However, the combination of A-1331852 and S63845 released all the BH3-only proteins from the anti-apoptotic counterparts and resulted in pronounced apoptosis (bold arrow). In the absence of all eight BH3-only proteins, neither A-1331852 nor S63845 alone resulted in apoptosis. However, a combination of A-1331852 and S63845 still resulted in pronounced apoptosis, even in the absence of all BH3-only proteins. This could be due to the release of another pro-apoptotic BH3 domain-containing ‘protein x’ from MCL-1 and/or BCL-XL, which in turn could activate BAX in the 8KO cells. Alternatively, BH3 mimetics could indirectly result in the accumulation of BAX (either by inhibition of retrotranslocation or by passive diffusion, following the neutralization of the anti-apoptotic members) in the outer mitochondrial membrane, which in turn could result in BAX activation and apoptosis in the 8KO cells
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