Novel Bcl-2 Inhibitors Selectively Disrupt the Autophagy-Specific Bcl-2-Beclin 1 Protein-Protein Interaction.

Autophagy plays essential roles in a wide variety of physiological processes, such as cellular homeostasis, metabolism, development, differentiation, and immunity. Selective pharmacological modulation of autophagy is considered a valuable potential therapeutic approach to treat diverse human diseases. However, development of such therapies has been greatly impeded by the lack of specific small molecule autophagy modulators. Here, we performed structure-activity relationship studies on a previously discovered weak Bcl-2 inhibitor SW076956, and developed a panel of small molecule compounds that selectively released Bcl-2-mediated inhibition of autophagy-related Beclin 1 compared to apoptosis-related Bax at nanomolar concentration. Our NMR analysis showed that compound 35 directly binds Bcl-2 and specifically inhibits the interaction between the Bcl-2 and Beclin 1 BH3 domains without disruption of the Bcl-2-Bax BH3 interaction. More broadly, this proof-of-concept study demonstrates that targeting protein-protein interactions of the intrinsic autophagy regulatory network can serve as a valuable strategy for the development of autophagy-based therapeutics.

Beth Levine's Legacy: From the Discovery of BECN1 to Therapies. A Mentees' Perspective.

With great sadness, the scientific community received the news of the loss of Beth Levine on 15 June 2020. Dr. Levine was a pioneer in the autophagy field and work in her lab led not only to a better understanding of the molecular mechanisms regulating the pathway, but also its implications in multiple physiological and pathological conditions, including its role in development, host defense, tumorigenesis, aging or metabolism. This review does not aim to provide a comprehensive view of autophagy, but rather an outline of some of the discoveries made by the group of Beth Levine, from the perspective of some of her own mentees, hoping to honor her legacy in science.

Mitophagy in aging and longevity.

The clearance of damaged or unwanted mitochondria by autophagy (also known as mitophagy) is a mitochondrial quality control mechanism postulated to play an essential role in cellular homeostasis, metabolism, and development and confers protection against a wide range of diseases. Proper removal of damaged or unwanted mitochondria is essential for organismal health. Defects in mitophagy are associated with Parkinson's, Alzheimer's disease, cancer, and other degenerative disorders. Mitochondria regulate organismal fitness and longevity via multiple pathways, including cellular senescence, stem cell function, inflammation, mitochondrial unfolded protein response (mtUPR), and bioenergetics. Thus, mitophagy is postulated to be pivotal for maintaining organismal healthspan and lifespan and the protection against aged-related degeneration. In this review, we will summarize recent understanding of the mechanism of mitophagy and aspects of mitochondrial functions. We will focus on mitochondria-related cellular processes that are linked to aging and examine current genetic evidence that supports the hypothesis that mitophagy is a pro-longevity mechanism.

Author Correction: Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.

High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor.

The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor suppression, and prevention of neurodegeneration and aging. However, the precise mechanisms of mitophagy remain uncertain. Here, we identify the inner mitochondrial membrane protein, prohibitin 2 (PHB2), as a crucial mitophagy receptor involved in targeting mitochondria for autophagic degradation. PHB2 binds the autophagosomal membrane-associated protein LC3 through an LC3-interaction region (LIR) domain upon mitochondrial depolarization and proteasome-dependent outer membrane rupture. PHB2 is required for Parkin-induced mitophagy in mammalian cells and for the clearance of paternal mitochondria after embryonic fertilization in C. elegans. Our findings pinpoint a conserved mechanism of eukaryotic mitophagy and demonstrate a function of prohibitin 2 that may underlie its roles in physiology, aging, and disease.

C. elegans SIRT6/7 homolog SIR-2.4 promotes DAF-16 relocalization and function during stress.

FoxO transcription factors and sirtuin family deacetylases regulate diverse biological processes, including stress responses and longevity. Here we show that the Caenorhabditis elegans sirtuin SIR-2.4--homolog of mammalian SIRT6 and SIRT7 proteins--promotes DAF-16-dependent transcription and stress-induced DAF-16 nuclear localization. SIR-2.4 is required for resistance to multiple stressors: heat shock, oxidative insult, and proteotoxicity. By contrast, SIR-2.4 is largely dispensable for DAF-16 nuclear localization and function in response to reduced insulin/IGF-1-like signaling. Although acetylation is known to regulate localization and activity of mammalian FoxO proteins, this modification has not been previously described on DAF-16. We find that DAF-16 is hyperacetylated in sir-2.4 mutants. Conversely, DAF-16 is acetylated by the acetyltransferase CBP-1, and DAF-16 is hypoacetylated and constitutively nuclear in response to cbp-1 inhibition. Surprisingly, a SIR-2.4 catalytic mutant efficiently rescues the DAF-16 localization defect in sir-2.4 null animals. Acetylation of DAF-16 by CBP-1 in vitro is inhibited by either wild-type or mutant SIR-2.4, suggesting that SIR-2.4 regulates DAF-16 acetylation indirectly, by preventing CBP-1-mediated acetylation under stress conditions. Taken together, our results identify SIR-2.4 as a critical regulator of DAF-16 specifically in the context of stress responses. Furthermore, they reveal a novel role for acetylation, modulated by the antagonistic activities of CBP-1 and SIR-2.4, in modulating DAF-16 localization and function.

HSF-1 regulators DDL-1/2 link insulin-like signaling to heat-shock responses and modulation of longevity.

Extended longevity is often correlated with increased resistance against various stressors. Insulin/IGF-1-like signaling (IIS) is known to have a conserved role in aging and cellular mechanisms against stress. In C. elegans, genetic studies suggest that heat-shock transcription factor HSF-1 is required for IIS to modulate longevity. Here, we report that the activity of HSF-1 is regulated by IIS. This regulation occurs at an early step of HSF-1 activation via two HSF-1 regulators, DDL-1 and DDL-2. Inhibition of DDL-1/2 increases longevity and thermotolerance in an hsf-1-dependent manner. Furthermore, biochemical analyses suggest that DDL-1/2 negatively regulate HSF-1 activity by forming a protein complex with HSF-1. The formation of this complex (DHIC) is affected by the phosphorylation status of DDL-1. Both the formation of DHIC and the phosphorylation of DDL-1 are controlled by IIS. Our findings point to DDL-1/2 as a link between IIS and the HSF-1 pathway.

Celecoxib extends C. elegans lifespan via inhibition of insulin-like signaling but not cyclooxygenase-2 activity.

One goal of aging research is to develop interventions that combat age-related illnesses and slow aging. Although numerous mutations have been shown to achieve this in various model organisms, only a handful of chemicals have been identified to slow aging. Here, we report that celecoxib, a nonsteroidal anti-inflammatory drug widely used to treat pain and inflammation, extends Caenorhabditis elegans lifespan and delays the age-associated physiological changes, such as motor activity decline. Celecoxib also delays the progression of age-related proteotoxicity as well as tumor growth in C. elegans. Celecoxib was originally developed as a potent cyclooxygenase-2 (COX-2) inhibitor. However, the result from a structural-activity analysis demonstrated that the antiaging effect of celecoxib might be independent of its COX-2 inhibitory activity, as analogs of celecoxib that lack COX-2 inhibitory activity produce a similar effect on lifespan. Furthermore, we found that celecoxib acts directly on 3'-phosphoinositide-dependent kinase-1, a component of the insulin/IGF-1 signaling cascade to increase lifespan.

Increased NBS1 expression is a marker of aggressive head and neck cancer and overexpression of NBS1 contributes to transformation.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) represents the sixth most frequent type of cancer worldwide. However, the molecular genetic alterations underlying its malignant behavior and progression are little known. We showed previously that c-MYC directly activates the expression of the DNA double-strand break repair gene NBS1, and NBS1 overexpression contributes to transformation. Here, we investigate the role of NBS1 overexpression in HNSCC. EXPERIMENTAL DESIGN: Immunohistochemistry analysis of NBS1 expression was done in 81 locally advanced HNSCC patients. Real-time PCR and Western blot analysis were used to confirm immunohistochemistry results. Human hypopharyngeal cancer cell lines (FADU) with overexpressing NBS1 (FADUNBS) or inducible short interference RNA to repress endogenous NBS1 (FADUNBSi) were generated by stable transfection. Soft agar clonogenicity assay was used to determine the transformation activity. Western blot analysis and phosphatidylinositol 3-kinase (PI3K) assay were done to evaluate the signaling pathways that were involved. RESULTS: NBS1 overexpression was identified in 45% of advanced HNSCC patients. It was an independent marker of poor prognosis. NBS1 expression levels correlated with the transformation activity of FADU clones and also correlated with the phosphorylation levels of Akt and its downstream target mammalian target of rapamycin (mTOR). PI3K activity was increased in NBS1-overexpressing FADU clones. NBS1 overexpression also correlated with increased Akt phosphorylation levels in tumor samples. CONCLUSIONS: Increased NBS1 expression is a significant prognostic marker of advanced HNSCC, and the underlying mechanism may involve the activation of the PI3K/Akt pathway.

Overexpression of NBS1 contributes to transformation through the activation of phosphatidylinositol 3-kinase/Akt.

Nijmegen breakage syndrome (NBS) is a chromosomal instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1 (p95) or nibrin, is a part of the hMre11 complex, a central player associated with double strand break repair. We previously demonstrated that c-Myc directly activates NBS1 expression. Here we have shown that constitutive expression of NBS1 in Rat1a and HeLa cells induces/enhances their transformation. Repression of endogenous NBS1 levels using short interference RNA reduces the transformation activity of two tumor cell lines. Increased NBS1 expression is observed in 40-52% of non-small cell lung carcinoma, hepatoma, and esophageal cancer samples. NBS1 overexpression stimulates phosphatidylinositol (PI) 3-kinase activity, leading to increased phosphorylation levels of Akt and its downstream targets such as glycogen synthase kinase 3beta and mammalian target of rapamycin in different cell lines and tumor samples. Transformation induced by NBS1 overexpression can be inhibited by a PI3-kinase inhibitor (LY294002). Repression of endogenous Akt expression by short interference RNA decreases the transformation activity of Rat1a cells overexpressing NBS1. These results indicate that overexpression of NBS1 is an oncogenic event that contributes to transformation through the activation of PI3-kinase/Akt.

Stimulation of c-Rel transcriptional activity by PKA catalytic subunit beta.

Nuclear factor kappaB (NF-kappaB) is a eukaryotic transcription factor which responds to different extracellular signals. It is involved in immune response, inflammation, and cell proliferation. Increased expression of c-Rel (or its viral homolog v-Rel), one component of the NF-kappaB factors, induces tumorigenesis in different systems. The activity of NF-kappaB can be regulated by protein kinase A (PKA) in a cAMP-independent manner. Our previous results showed that c-MYC induces the activity of PKA by inducing the transcription of the gene encoding the PKA catalytic subunit beta (PKA-Cbeta). Constitutive expression of PKA-Cbeta in Rat1a cells induces their transformation. Here we show that CREB is unlikely to be a phosphorylation target of PKA-Cbeta as characterized by different cell lines. Electrophoretic mobility shift assays showed that c-Rel is present as a significant component of the NF-kappaB factors in c-MYC overexpressing status. The transcriptional activity of c-Rel was significantly stimulated by PKA-Cbeta. Coactivators p300/CBP are at least partially responsible for the enhanced activation mediated by c-Rel and PKA-Cbeta. Interaction between c-Rel and PKA-Cbeta was demonstrated using coimmunoprecipitation assays. Immunoprecipitation-in vitro phosphorylation assays showed the direct phosphorylation of c-Rel by PKA-Cbeta. These results indicate that c-Rel is a reasonable phosphorylation target of PKA-Cbeta, and that the transcriptional activity of c-Rel is stimulated by PKA-Cbeta possibly through the interaction with p300/CBP.