Yogesh Tengarai Ganesan

An interconnected hierarchical model of cell death regulation by the BCL-2 family

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.

PUMA and BIM Are Required for Oncogene Inactivation–Induced Apoptosis

Combining drugs that inhibit antiapoptotic proteins with oncogenic kinase inhibitors may overcome drug resistance in cancer therapy. Using Addiction Against Cancer Oncogene-addicted cancer cells depend on a particular oncogenic protein for survival and die when the oncogenic protein is inactivated. For example, lung cancers with an abnormally active form of the epidermal growth factor receptor (EGFR) and breast cancers with amplification of the gene encoding the human epidermal growth factor receptor 2 (HER2) regress when exposed to drugs called tyrosine kinase inhibitors that block the activity of these receptors. Bean et al. analyzed breast and lung cancer cell lines treated with tyrosine kinase inhibitors and mice in which EGFRs were inactivated by genetic ablation. They found that two signaling pathways, the phosphoinositide 3-kinase (PI3K)–AKT and mitogen-activated or extracellular signal–regulated protein kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) pathways, were inactivated, resulting in an increase in the abundance of PUMA and BIM, two proteins that promote cell death. Furthermore, cancer cell lines resistant to tyrosine kinase inhibitors were killed by the combination of a PI3K inhibitor and a drug that inhibits antiapoptotic proteins. Thus, pharmacologically enhancing the death pathway may overcome or prevent resistance to tyrosine kinase inhibitors. 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 EGFRL858R-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.

Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy

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-XL or MCL-1, which correlated with the respective protein expression ratio. ABT-263 (navitoclax), a BCL-2/BCL-XL inhibitor, prevented BCL-XL from sequestering activator BH3-only molecules (BH3s) and BAX but not BAK. Consequently, ABT-263 failed to kill BCL-XL-addicted cells with low activator BH3s and BCL-XL 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-XL, 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.

An Autoinhibited Dimeric Form of BAX Regulates the BAX Activation Pathway.

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.

Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737

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.

A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

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.

ΔNp63 Inhibits Oxidative Stress-Induced Cell Death, Including Ferroptosis, and Cooperates with the BCL-2 Family to Promote Clonogenic Survival

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.

Abstract 3555: Targeting the BCL-2 family in small cell lung caner

Small cell lung cancer (SCLC) represents 13% of all lung cancer cases, affecting approximately 30,000 people annually in the United States. The prognosis for patients with SCLC is poor, and cancer-specific mortality of this malignancy is 95% at five years. This dismal prognosis has been further marred by the absence of major improvements in its treatment: there have been no substantial changes in the standard of care for advanced SCLC over the last three decades. Interestingly, small molecule inhibitors of anti-apoptotic BCL-2 family members BCL-2 and BCL-XL, ABT-737 and its orally bioavailable analog ABT-263, were shown to effectively kill some SCLC cell lines in preclinical studies, suggesting that targeting the BCL-2 family proteins may hold promise for the treatment of this dreadful cancer. However, these inhibitors do not inhibit MCL-1, another anti-apoptotic BCL-2 family member, which may explain why multiple clinical trials have not led to meaningful results. Because there are no effective MCL-1 inhibitors developed yet, we propose to identify the best strategy that enhances the apoptotic effect of ABT-737/263. To this end, we have performed a high-throughput screen in ABT-737/263 resistant H196 cell lines using two libraries, a library of FDA-approved anti-cancer agents and a pathway inhibitor library including ∼1000 compounds. As a result, doxorubicin and dinaciclib were identified as synergizers of ABT-737/263 in triggering apoptosis. These two drugs enhanced the death inducing activity of ABT-737/263 through downregulation of MCL-1 mRNA. Interestingly, some of the ABT-737/263 resistant cell lines rely on MCL-1 for survival and thereby were killed by dinaciclib or doxorubicin as a single agent, indicating that predictive biomarkers of cellular addiction to anti-apoptotic BCL-2 family proteins (BCL-2s) can guide treatment decisions and reduce unwanted toxicity. Importantly, we revealed that the expression ratio between BCL-2, BCL-XL, and MCL-1 could predict cellular addiction to BCL-2s. Surprisingly, we demonstrated that BCL-XL overexpression is an another potential mechanism behind ABT-263 resistance. ABT-263 failed to inhibit the interaction between BCL-XL and BAX/BAK in the absence of activator BH3-only molecules and thereby failed to kill one BCL-XL-addicted SCLC cell line with low expression of activator BH3-only molecules. Finally, we showed that BCL-2 selective inhibitor ABT-199 can also cooperate with doxorubicin or dinaciclib to kill ABT-263-resistant cell lines. The in vivo efficacy of combination therapy including ABT-199 and doxorubicin was demonstrated in a SCLC xenograft model. These data have direct translational implications for the treatment of SCLC. Citation Format: Akane Inoue-Yamauchi, Paul Jeng, Kwanghee Kim, Hui-Chen Chen, Song Han, Yogesh Tengarai Ganesan, Yiyu Dong, Sylvia Jebiwott, James J. Hsieh, Emily H. Cheng. Targeting the BCL-2 family in small cell lung caner. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3555.