Differential regulation of BAX and BAK apoptotic activity revealed by small molecules

Abstract

Defective apoptosis mediated by B cell lymphoma 2 antagonist/killer (BAK) or B cell lymphoma 2-associated X protein (BAX) underlies various pathologies including autoimmune and degenerative conditions. On mitochondria, voltage-dependent anion channel 2 (VDAC2) interacts with BAK and BAX through a common interface to inhibit BAK or to facilitate BAX apoptotic activity. We identified a small molecule (WEHI-3773) that inhibits interaction between VDAC2 and BAK or BAX revealing contrasting effects on their apoptotic activity. WEHI-3773 inhibits apoptosis mediated by BAX by blocking VDAC2-mediated BAX recruitment to mitochondria. Conversely, WEHI-3773 promotes BAK-mediated apoptosis by limiting inhibitory sequestration by VDAC2. In cells expressing both pro-apoptotic proteins, apoptosis promotion by WEHI-3773 dominates, because activated BAK activates BAX through a feed-forward mechanism. Loss of BAX drives resistance to the BCL-2 inhibitor venetoclax in some leukemias. WEHI-3773 overcomes this resistance by promoting BAK-mediated killing. This work highlights the coordination of BAX and BAK apoptotic activity through interaction with VDAC2 that may be targeted therapeutically.

Fig. 1.. High-throughput screening identifies inhibitors of BAX-driven apoptosis.

(A) Experimental workflow and selection criteria used for the discovery of inhibitors of human BAX–driven apoptosis. (B) Schematics of assays measuring different steps in the apoptosis pathway. (C) Dose-response curves for 17 hit compounds tested on mitochondria in the presence of BIM-BH3 and measured using JC-1 fluorescence assay. WEHI-9625 serves as a control (25). Data are from the validation screening cascade, a single experiment. (D) Structure of compound 1. (E) Dose-response curve for compound 1 tested in the primary screening assay using CellTiter-Glo. Data are from two independent experiments. (F) Structure of WEHI-3773. (G) Dose-response curve for WEHI-3773 tested in the primary screening assay using CellTiter-Glo. Data are from two independent experiments. (H) Dose-response viability curves of KMS-12-PE in the presence of ABT-737, measured by PI uptake and flow cytometry. Data are the means ± SEM of three independent experiments. (I) Impact of WEHI-3773 on mitochondrial membrane potential measured by JC-1 staining and flow cytometry in the presence of human BIM-BH3 peptides. Data are the means ± SEM of three independent experiments.

Fig. 2.. WEHI-3773 inhibits BAX activation and translocation to limit MOMP and protect cells.

(A) WEHI-3773’s dose-dependent inhibition of cell death in BAK^−/− HeLa treated with ABT-737 and S63845, measured by PI uptake and flow cytometry. Data are the means ± SEM of three independent experiments. (B) WEHI-3773’s dose-dependent inhibition of mitochondrial depolarization induced by the BIM-BH3 peptide. Depolarization was measured by JC-1 staining and flow cytometry. Data are the means ± SEM of three independent experiments. (C) Representative images of colony formation assay using BAK^−/− HeLa after 5 days of culture. (D) Quantification of colony number. Data are the means and SEM of three independent experiments. (E) Intracellular staining and flow cytometry to measure conformationally activated BAX and cytochrome c in BAK^−/− HeLa. (F) Western blot analysis of BAK^−/− HeLa treated with WEHI-3773, ABT-737, and S63845 at indicated concentrations for 2 hours. Representative blots from n = 3 independent experiments are shown. Cytosol and membrane fractions are from the same blot (see the Supplementary Materials) but have been separated for ease of comparison.

Fig. 3.. WEHI-3773 inhibits VDAC2-mediated BAX recruitment to mitochondria.

(A) Western blot analysis of Vdac2^−/− or Vdac2^+/+ MEFs treated with WEHI-3773 alone at indicated concentrations for 2 hours. Representative blots of at least three independent experiments are shown. Cytosol and membrane fractions for each genotype are from the same blot (see the Supplementary Materials) but have been separated for ease of comparison. (B) Dose-response viability curves of Bak^−/− MEFs measured by PI uptake and flow cytometry. Cells were pretreated with WEHI-3773 for 2 hours first, and then EC₇₀ concentrations of ABT-737 and S63845 were added for another 22 hours before measurement. Data are the means ± SEM of three independent experiments. (C) Western blot (WB) analysis of BAK^−/− mitochondria treated with WEHI-3773 or human BIM-BH3 peptide at indicated concentrations for 30 min, followed by BN-PAGE. Representative blots of at least three independent experiments are shown. (D) Proposed working model showing that WEHI-3773 acts on inhibiting BAX:VDAC2 interaction on mitochondria (gray) to limit MOMP and release of mitochondrial factors (brown dots).

Fig. 4.. WEHI-3773 potentiates BAK activation by destabilizing the BAK:VDAC2 interaction.

(A and B) WEHI-3773’s potentiation of cell death combined with BH3 mimetics, measured by PI uptake and flow cytometry. Data are the means ± SEM of three independent experiments. (C) Impact of WEHI-3773 on mitochondrial membrane potential measured by JC-1 staining and flow cytometry. Means ± SEM of three independent experiments. (D) Intracellular staining and flow cytometry to measure activated BAK and preserved cytochrome c level within BAX^−/− HeLa. (E) WEHI-3773’s impact on cell death in MEFs, measured by PI uptake and flow cytometry. Cells were pretreated with WEHI-3773 for 2 hours, and then ABT-737 and S63845 (2.4 nM for Vdac2^−/− Bax^−/− MEFs and 40 nM for Vdac2^+/+ Bax^−/− MEFs) were added for another 22 hours. Means ± SEM of three independent experiments. (F) BN-PAGE of BAX^−/− HeLa treated with compounds for 2 hours. Representative blots of three independent experiments are shown. (G) Dose response in MEFs re-expressing indicated BAX variants, measured by PI uptake and flow cytometry. Means ± SEM of three independent experiments. (H) Schematic model for WEHI-3773 on mitochondria (gray) to potentiate MOMP and release of mitochondrial factors (brown dots).

Fig. 5.. The β7-to-β10 region of VDAC2 mediates the action of WEHI-3773.

(A and B) Viability of KMS-12-PE cells treated for 48 hours, measured by PI uptake and flow cytometry. Data are the means ± SEM of three independent experiments. (C) Immunoblots of mitochondria treated with WEHI-3773 for 30 min, followed by addition of proteinase K. Representative blots of three independent experiments are shown. (D) Immunoblots of (C) were quantified and normalized to samples in the second lane. Data are the means ± SEM of three independent experiments. (E) Western blot of recombinant proteins treated with WEHI-3773 for 30 min, followed by addition of proteinase K. Representative blots of four independent experiments are shown. (F) Immunoblots of (E) were quantified and normalized to samples in the second lane. Data are the means ± SEM of four independent experiments. (G) Schematic representation of VDAC1/VDAC2 chimeric constructs and the predicted VDAC2 structure (AF-P45880-F1) (, 53). VDAC2 is presented as cyan cartoons with the region examined in this study colored gold. IMS, intermembrane space. (H) WEHI-3773’s impact on cell death in MEFs expressing indicated constructs, measured by PI uptake and flow cytometry. Cells were pretreated with WEHI-3773 for 2 hours, and then ABT-737 and S63845 were added for another 22 hours. Data are the means ± SEM of three independent experiments.

Fig. 6.. WEHI-3773 sensitizes BAX-mutant leukemia cells to BH3 mimetics.

(A) Western blot of lysates from MV4;11 cells transduced with an empty vector or BAX guide RNA. (B) BAX^−/− MV4;11 cells were treated with the indicated concentration of BH3 mimetics with or without WEHI-3773 (200 nM) before the assessment of cell viability after 48 hours. (C to E) BAX^−/− MV4;11 cells were treated with the indicated concentration of venetoclax (C), S63845 (D), or a combination of venetoclax and S63845 (E) with or without WEHI-3773 before the assessment of cell viability after 48 hours. Data are the means ± SEM of three independent experiments. Two-way analysis of variance (ANOVA) with Tukey test; **P < 0.01 and ***P < 0.001. (F to H) OCI-AML3 cells rendered resistant to venetoclax (BCL-2i R) were treated with the indicated concentration of venetoclax (F), S63845 (G), or a combination of venetoclax and S63845 (H) with or without WEHI-3773 before the assessment of cell viability after 48 hours. Data are means ± SEM of three independent experiments. Two-way ANOVA with Tukey test; *P < 0.05, **P < 0.01, and ***P < 0.001.