Pro-apoptotic BAX is a cell fate regulator playing an important role in cellular homeostasis and pathological cell death. BAX is predominantly localized in the cytosol, where it has a quiescent ...monomer conformation. Following a pro-apoptotic trigger, cytosolic BAX is activated and translocates to the mitochondria to initiate mitochondrial dysfunction and apoptosis. Here, cellular, biochemical, and structural data unexpectedly demonstrate that cytosolic BAX also has an inactive dimer conformation that regulates its activation. The full-length crystal structure of the inactive BAX dimer revealed an asymmetric interaction consistent with inhibition of the N-terminal conformational change of one protomer and the displacement of the C-terminal helix α9 of the second protomer. This autoinhibited BAX dimer dissociates to BAX monomers before BAX can be activated. Our data support a model whereby the degree of apoptosis induction is regulated by the conformation of cytosolic BAX and identify an unprecedented mechanism of cytosolic BAX inhibition.
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•BAX forms inactive dimers in the cytosol•Structure of the BAX dimer reveals a novel autoinhibition mechanism•Inactive BAX dimer dissociates to BAX monomers before BAX is activated•Autoinhibited dimer regulates BAX activation and BAX-mediated apoptosis
Cytosolic BAX is thought to be a monomer that is activated by BH3-only proteins to induce cell death. Garner et al. show that cytosolic BAX can form a homo-dimer whose structure inhibits BAX activation, revealing a step in the regulation of BAX that modulates apoptosis.
Multidomain pro-apoptotic BAX and BAK, once activated, permeabilize mitochondria to trigger apoptosis, whereas anti-apoptotic BCL-2 members preserve mitochondrial integrity. The BH3-only molecules ...(BH3s) promote apoptosis by either activating BAX-BAK or inactivating anti-apoptotic members. Here, we present biochemical and genetic evidence that NOXA is a bona fide activator BH3. Using combinatorial gain-of-function and loss-of-function approaches in Bid(-/-)Bim(-/-)Puma(-/-)Noxa(-/-) and Bax(-/-)Bak(-/-) cells, we have constructed an interconnected hierarchical model that accommodates and explains how the intricate interplays between the BCL-2 members dictate cellular survival versus death. BID, BIM, PUMA and NOXA directly induce stepwise, bimodal activation of BAX-BAK. BCL-2, BCL-XL and MCL-1 inhibit both modes of BAX-BAK activation by sequestering activator BH3s and 'BH3-exposed' monomers of BAX-BAK, respectively. Furthermore, autoactivation of BAX and BAK can occur independently of activator BH3s through downregulation of BCL-2, BCL-XL and MCL-1. Our studies lay a foundation for targeting the BCL-2 family for treating diseases with dysregulated apoptosis.
BCL-2 family proteins are central regulators of mitochondrial apoptosis and validated anti-cancer targets. Using small cell lung cancer (SCLC) as a model, we demonstrated the presence of differential ...addiction of cancer cells to anti-apoptotic BCL-2, BCL-X
or MCL-1, which correlated with the respective protein expression ratio. ABT-263 (navitoclax), a BCL-2/BCL-X
inhibitor, prevented BCL-X
from sequestering activator BH3-only molecules (BH3s) and BAX but not BAK. Consequently, ABT-263 failed to kill BCL-X
-addicted cells with low activator BH3s and BCL-X
overabundance conferred resistance to ABT-263. High-throughput screening identified anthracyclines including doxorubicin and CDK9 inhibitors including dinaciclib that synergized with ABT-263 through downregulation of MCL-1. As doxorubicin and dinaciclib also reduced BCL-X
, the combinations of BCL-2 inhibitor ABT-199 (venetoclax) with doxorubicin or dinaciclib provided effective therapeutic strategies for SCLC. Altogether, our study highlights the need for mechanism-guided targeting of anti-apoptotic BCL-2 proteins to effectively activate the mitochondrial cell death programme to kill cancer cells.
The BCL-2 family proteins are central regulators of apoptosis. However, cells deficient for BAX and BAK or overexpressing BCL-2 still succumb to oxidative stress upon DNA damage or matrix detachment. ...Here, we show that ΔNp63α overexpression protects cells from oxidative stress induced by oxidants, DNA damage, anoikis, or ferroptosis-inducing agents. Conversely, ΔNp63α deficiency increases oxidative stress. Mechanistically, ΔNp63α orchestrates redox homeostasis through transcriptional control of glutathione biogenesis, utilization, and regeneration. Analysis of a lung squamous cell carcinoma dataset from The Cancer Genome Atlas (TCGA) reveals that TP63 amplification/overexpression upregulates the glutathione metabolism pathway in primary human tumors. Strikingly, overexpression of ΔNp63α promotes clonogenic survival of p53−/−Bax−/−Bak−/− cells against DNA damage. Furthermore, co-expression of BCL-2 and ΔNp63α confers clonogenic survival against matrix detachment, disrupts the luminal clearance of mammary acini, and promotes cancer metastasis. Our findings highlight the need for a simultaneous blockade of apoptosis and oxidative stress to promote long-term cellular well-being.
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•ΔNp63α is a key cellular guardian against oxidative stress, including ferroptosis•ΔNp63α inhibits oxidative stress through regulation of glutathione metabolism•ΔNp63α cooperates with the BCL-2 family to promote long-term clonogenic survival•TP63 amplification upregulates glutathione metabolism to promote tumorigenesis
Apoptosis-defective cells remain vulnerable to oxidative stress, which limits long-term survival. Wang et al. identify ΔNp63α as a central regulator of redox homeostasis through transcriptional control of a tightly coupled glutathione metabolic circuit. ΔNp63α alleviates oxidative stress and cooperates with the BCL-2 family to promote both long-term cellular well-being and cancer metastasis.
The clinical efficacy of tyrosine kinase inhibitors supports the dependence of distinct subsets of cancers on specific driver mutations for survival, a phenomenon called "oncogene addiction." We ...demonstrate that PUMA and BIM are the key apoptotic effectors of tyrosine kinase inhibitors in breast cancers with amplification of the gene encoding human epidermal growth factor receptor 2 (HER2) and lung cancers with epidermal growth factor receptor (EGFR) mutants. The BH3 domain containing proteins BIM and PUMA can directly activate the proapoptotic proteins BAX and BAK to permeabilize mitochondria, leading to caspase activation and apoptosis. We delineated the signal transduction pathways leading to the induction of BIM and PUMA by tyrosine kinase inhibitors. Inhibition of the mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway caused increased abundance of BIM, whereas antagonizing the phosphoinositide 3-kinase (PI3K)-AKT pathway triggered nuclear translocation of the FOXO transcription factors, which directly activated the PUMA promoter. In a mouse breast tumor model, the abundance of PUMA and BIM was increased after inactivation of HER2. Moreover, deficiency of Bim or Puma impaired caspase activation and reduced tumor regression caused by inactivation of HER2. Similarly, deficiency of Puma impeded the regression of EGFR(L858R)-driven mouse lung tumors upon inactivation of the EGFR-activating mutant. Overall, our study identified PUMA and BIM as the sentinels that interconnect kinase signaling networks and the mitochondrion-dependent apoptotic program, which offers therapeutic insights for designing novel cell death mechanism-based anticancer strategies.
Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax ...N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer.
The clinical success of EGFR inhibitors in EGFR-mutant lung cancer is limited by the eventual development of acquired resistance. We hypothesize that enhancing apoptosis through combination therapies ...can eradicate cancer cells and reduce the emergence of drug-tolerant persisters. Through high-throughput screening of a custom library of ∼1,000 compounds, we discover Aurora B kinase inhibitors as potent enhancers of osimertinib-induced apoptosis. Mechanistically, Aurora B inhibition stabilizes BIM through reduced Ser87 phosphorylation, and transactivates PUMA through FOXO1/3. Importantly, osimertinib resistance caused by epithelial-mesenchymal transition (EMT) activates the ATR-CHK1-Aurora B signaling cascade and thereby engenders hypersensitivity to respective kinase inhibitors by activating BIM-mediated mitotic catastrophe. Combined inhibition of EGFR and Aurora B not only efficiently eliminates cancer cells but also overcomes resistance beyond EMT.
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•Aurora kinase inhibitors combined with osimertinib prevent and overcome resistance•Concurrent inhibition of EGFR and AURKB maximizes BIM- and PUMA-mediated apoptosis•EMT activates ATR-CHK1-AURKB and sensitizes cancer cells to these kinase inhibitors•Mitotic catastrophe triggered by ATR-CHK1-AURKB inhibitors involves BIM and BAX/BAK
Tanaka et al. identify Aurora kinase inhibitors as potent enhancers of osimertinib-induced apoptosis by HTS. Concurrent inhibition of EGFR and AURKB maximizes BIM- and PUMA-mediated apoptosis to eradicate cancer cells. Osimertinib resistance caused by EMT activates ATR-CHK1-AURKB and engenders hypersensitivity to these kinase inhibitors by activating BIM-mediated mitotic catastrophe.
Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the ...retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release.
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