ABSTRACT
In stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2-α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other's surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis.
Subject(s)
Apoptosis , Crystallography, X-Ray , bcl-2-Associated X Protein/chemistry , Amino Acid Sequence , Animals , Cytochromes c/metabolism , Dimerization , Embryo, Mammalian/metabolism , Hydrophobic and Hydrophilic Interactions , Liposomes/metabolism , Liver/metabolism , Mice , Mitochondria/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Tertiary , Sequence Alignment , bcl-2-Associated X Protein/metabolismABSTRACT
Certain BH3-only proteins transiently bind and activate Bak and Bax, initiating their oligomerization and the permeabilization of the mitochondrial outer membrane, a pivotal step in the mitochondrial pathway to apoptosis. Here we describe the first crystal structures of an activator BH3 peptide bound to Bak and illustrate their use in the design of BH3 derivatives capable of inhibiting human Bak on mitochondria. These BH3 derivatives compete for the activation site at the canonical groove, are the first engineered inhibitors of Bak activation, and support the role of key conformational transitions associated with Bak activation.
Subject(s)
Apoptosis/drug effects , Bcl-2-Like Protein 11 , Mitochondria , Peptides , bcl-2 Homologous Antagonist-Killer Protein , Animals , Bcl-2-Like Protein 11/chemistry , Bcl-2-Like Protein 11/pharmacology , Cell Line, Transformed , Humans , Mice , Mitochondria/genetics , Mitochondria/metabolism , Peptides/chemistry , Peptides/pharmacology , Protein Binding , Structure-Activity Relationship , bcl-2 Homologous Antagonist-Killer Protein/chemistry , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.
Subject(s)
Apoptosis/genetics , Blood Platelets/cytology , T-Lymphocytes/cytology , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blood Platelets/drug effects , Blood Platelets/metabolism , Cell Line , Cell Survival/genetics , Humans , Mice , Mice, Inbred C57BL , Mutation , Protein Binding , Protein Conformation , Protein Domains/genetics , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , bcl-2 Homologous Antagonist-Killer Protein/geneticsABSTRACT
Landmark genome-wide association studies (GWAS) identified that mutations in autophagy genes correlated with inflammatory bowel disease (IBD), a heterogenous disease characterised by prolonged inflammation of the gastrointestinal tract, that can reduce a person's quality of life. Autophagy, the delivery of intracellular components to the lysosome for degradation, is a critical cellular housekeeping process that removes damaged proteins and turns over organelles, recycling their amino acids and other constituents to supply cells with energy and necessary building blocks. This occurs under both basal and challenging conditions such as nutrient deprivation. An understanding of the relationship between autophagy, intestinal health and IBD aetiology has improved over time, with autophagy having a verified role in the intestinal epithelium and immune cells. Here, we discuss research that has led to an understanding that autophagy genes, including ATG16L, ATG5, ATG7, IRGM, and Class III PI3K complex members, contribute to innate immune defence in intestinal epithelial cells (IECs) via selective autophagy of bacteria (xenophagy), how autophagy contributes to the regulation of the intestinal barrier via cell junctional proteins, and the critical role of autophagy genes in intestinal epithelial secretory subpopulations, namely Paneth and goblet cells. We also discuss how intestinal stem cells can utilise autophagy. Importantly, mouse studies have provided evidence that autophagy deregulation has serious physiological consequences including IEC death and intestinal inflammation. Thus, autophagy is now established as a key regulator of intestinal homeostasis. Further research into how its cytoprotective mechanisms can prevent intestinal inflammation may provide insights into the effective management of IBD.
Subject(s)
Genome-Wide Association Study , Inflammatory Bowel Diseases , Animals , Mice , Quality of Life , Epithelial Cells/metabolism , Intestinal Mucosa , Inflammation/metabolism , Autophagy/geneticsABSTRACT
The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins. Inhibition of the anti-apoptotic members represents a strategy to induce apoptotic cell death in cancer cells. We have measured the membrane binding properties of a series of peptides, including modified α/ß-peptides, designed to exhibit enhanced membrane permeability to allow cell entry and improved access for engagement of Bcl-2 family members. The peptide cargo is based on the pro-apoptotic protein Bim, which interacts with all anti-apoptotic proteins to initiate apoptosis. The α/ß-peptides contained cyclic ß-amino acid residues designed to increase their stability and membrane-permeability. Dual polarisation interferometry was used to study the binding of each peptide to two different model membrane systems designed to mimic either the plasma membrane or the outer mitochondrial membrane. The impact of each peptide on the model membrane structure was also investigated, and the results demonstrated that the modified peptides had increased affinity for the mitochondrial membrane and significantly altered the structure of the bilayer. The results also showed that the presence of an RRR motif significantly enhanced the ability of the peptides to bind to and insert into the mitochondrial membrane mimic, and provide insights into the role of selective membrane targeting of peptides.
ABSTRACT
The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.
Subject(s)
Lymphoma/genetics , Lymphoma/therapy , Mutation , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Protein p53/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , Gene Expression Regulation, Neoplastic , Humans , Lymphoma/pathology , Mice , Mice, Inbred C57BL , Protein Binding , Proto-Oncogene Proteins c-myc/geneticsABSTRACT
The deregulation of apoptosis is a key contributor to tumourigenesis as it can lead to the unwanted survival of rogue cells. Drugs known as the BH3-mimetics targeting the pro-survival members of the BCL-2 protein family to induce apoptosis in cancer cells have achieved clinical success for the treatment of haematological malignancies. However, despite our increasing knowledge of the pro-survival factors mediating the unwanted survival of solid tumour cells, and our growing BH3-mimetics armamentarium, the application of BH3-mimetic therapy in solid cancers has not reached its full potential. This is mainly attributed to the need to identify clinically safe, yet effective, combination strategies to target the multiple pro-survival proteins that typically mediate the survival of solid tumours. In this review, we discuss current and exciting new developments in the field that has the potential to unleash the full power of BH3-mimetic therapy to treat currently recalcitrant solid malignancies.
Subject(s)
Apoptosis , Neoplasms/drug therapy , Proto-Oncogene Proteins c-bcl-2/metabolism , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Humans , Neoplasms/metabolismABSTRACT
The discovery of a new class of small molecule compounds that target the BCL-2 family of anti-apoptotic proteins is one of the great success stories of basic science leading to translational outcomes in the last 30 years. The eponymous BCL-2 protein was identified over 30 years ago due to its association with cancer. However, it was the unveiling of the biochemistry and structural biology behind it and its close relatives' mechanism(s)-of-action that provided the inspiration for what are now known as 'BH3-mimetics', the first clinically approved drugs designed to specifically inhibit protein-protein interactions. Herein, we chart the history of how these drugs were discovered, their evolution and application in cancer treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Delivery Systems , Drug Discovery , Proto-Oncogene Proteins c-bcl-2/drug effects , Apoptosis/drug effects , Humans , Ligands , Molecular MimicryABSTRACT
Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.
Subject(s)
Apoptosis/physiology , Small Molecule Libraries/metabolism , Voltage-Dependent Anion Channel 2/physiology , bcl-2 Homologous Antagonist-Killer Protein/physiology , Animals , Mice , Protein Binding , Voltage-Dependent Anion Channel 2/metabolismABSTRACT
Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival , Gene Deletion , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred C57BL , Myeloid Cell Leukemia Sequence 1 Protein , Tamoxifen/pharmacology , Tumor Cells, CulturedABSTRACT
Interactions between the pro-survival and pro-apoptotic members of the Bcl-2 family of proteins dictate whether a cell lives or dies. Much of our knowledge of the molecular details of these interactions has come from biochemical and structural studies on the pro-survival protein Bcl-xL. The first high-resolution structure of any Bcl-2 family member was of Bcl-xL, which revealed the conserved topology amongst all family members. Subsequent structures of Bcl-xL complexes with pro-apoptotic ligands demonstrated the general features of all pro-survival:pro-apoptotic complexes. Structural studies involving Bcl-xL were also the basis for the discovery of the first small-molecule pro-survival protein inhibitors, leading ultimately to the development of a new class of drugs now successfully used for cancer treatment in the clinic. This article will review our current knowledge of the structural biology of Bcl-xL and how this has impacted our understanding of the molecular details of the intrinsic apoptotic pathway.
Subject(s)
bcl-X Protein/chemistry , Animals , Binding Sites , Humans , Protein Binding , Protein Multimerization , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , bcl-X Protein/metabolismABSTRACT
Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.
Subject(s)
Apoptosis , Dendritic Cells/cytology , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Cell Separation , Cell Survival , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Signal Transduction , Spleen/immunology , Spleen/metabolism , T-Lymphocytes/cytology , Transgenes , bcl-X Protein/metabolismABSTRACT
Beclin 1 is a 450 amino acid protein that plays critical roles in the early stages of autophagosome formation. We recently reported the successful expression, purification and structural characterisation of the entire N-terminal region of Beclin 1 (residues 1-150), including its backbone NMR chemical shift assignments. Based on assigned backbone NMR chemical shifts, it has been established that the N-terminal region of Beclin 1 (1-150), including the BH3 domain (112-123), is intrinsically disordered in the absence of its interaction partners. Here, a detailed study of its conformational preference and backbone dynamics obtained from an analysis of its secondary structure populations using the δ2D method, and the measurements of effective hydrodynamic radius as well as (1)H temperature coefficients, (1)H solvent exchange rates, and (15)N relaxation parameters of backbone amides using NMR spectroscopy is reported. These data provide further evidence for the intrinsically disordered nature of the N-terminal region of Beclin 1 and support the view that the helical conformation adopted by the Beclin 1 BH3 domain upon interaction with binding partners such as BCL-2 pro-survival proteins is likely induced rather than pre-existing.
Subject(s)
Beclin-1/chemistry , Intrinsically Disordered Proteins/chemistry , Humans , Kinetics , Magnetic Resonance Spectroscopy , Nitrogen Isotopes , Protein Conformation, alpha-Helical , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Staining and Labeling/methods , ThermodynamicsABSTRACT
Bcl-2 homology 3 (BH3) domains are short sequence motifs that mediate nearly all protein-protein interactions between B cell lymphoma 2 (Bcl-2) family proteins in the intrinsic apoptotic cell death pathway. These sequences are found on both pro-survival and pro-apoptotic members, although their primary function is believed to be associated with induction of cell death. Here, we identify critical features of the BH3 domains of pro-survival proteins that distinguish them functionally from their pro-apoptotic counterparts. Biochemical and x-ray crystallographic studies demonstrate that these differences reduce the capacity of most pro-survival proteins to form high affinity "BH3-in-groove" complexes that are critical for cell death induction. Switching these residues for the corresponding residues in Bcl-2 homologous antagonist/killer (Bak) increases the binding affinity of isolated BH3 domains for pro-survival proteins; however, their exchange in the context of the parental protein causes rapid proteasomal degradation due to protein destabilization. This is supported by further x-ray crystallographic studies that capture elements of this destabilization in one pro-survival protein, Bcl-w. In pro-apoptotic Bak, we demonstrate that the corresponding distinguishing residues are important for its cell-killing capacity and antagonism by pro-survival proteins.
Subject(s)
Apoptosis Regulatory Proteins/chemistry , bcl-X Protein/chemistry , Amino Acid Sequence , Animals , Apoptosis , Apoptosis Regulatory Proteins/physiology , Cell Survival , Cells, Cultured , Crystallography, X-Ray , Cytochromes c/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Stability , bcl-X Protein/physiologyABSTRACT
Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and ß-amino acid residues ("α/ß-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/ß-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/ß-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/ß-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/ß-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.
Subject(s)
Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Intracellular Space/drug effects , Intracellular Space/metabolism , Protein Folding , Amino Acid Sequence , Animals , Apoptosis Regulatory Proteins/chemistry , Bcl-2-Like Protein 11 , Cell Membrane Permeability , Cell Survival/drug effects , Cell-Penetrating Peptides/metabolism , Cytochromes c/metabolism , HCT116 Cells , Humans , Membrane Proteins/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Peptide Hydrolases/metabolism , Protein Binding/drug effects , Protein Stability , Protein Structure, Tertiary , Proteolysis , Proto-Oncogene Proteins/chemistryABSTRACT
Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.
Subject(s)
Apoptosis , Cell Membrane/metabolism , Fas Ligand Protein/metabolism , fas Receptor/metabolism , Animals , Antibodies, Antinuclear/immunology , Cytidine Deaminase/metabolism , Cytotoxicity, Immunologic , Fas Ligand Protein/deficiency , Fas Ligand Protein/genetics , Glomerulonephritis/metabolism , Histiocytic Sarcoma/metabolism , Hypergammaglobulinemia/metabolism , Lupus Erythematosus, Systemic/metabolism , Lymphatic Diseases/metabolism , Mice , Mice, Inbred C57BL , Mutation , Splenomegaly/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Anorexia and weight loss are part of the wasting syndrome of late-stage cancer, are a major cause of morbidity and mortality in cancer, and are thought to be cytokine mediated. Macrophage inhibitory cytokine-1 (MIC-1) is produced by many cancers. Examination of sera from individuals with advanced prostate cancer showed a direct relationship between MIC-1 abundance and cancer-associated weight loss. In mice with xenografted prostate tumors, elevated MIC-1 levels were also associated with marked weight, fat and lean tissue loss that was mediated by decreased food intake and was reversed by administration of antibody to MIC-1. Additionally, normal mice given systemic MIC-1 and transgenic mice overexpressing MIC-1 showed hypophagia and reduced body weight. MIC-1 mediates its effects by central mechanisms that implicate the hypothalamic transforming growth factor-beta receptor II, extracellular signal-regulated kinases 1 and 2, signal transducer and activator of transcription-3, neuropeptide Y and pro-opiomelanocortin. Thus, MIC-1 is a newly defined central regulator of appetite and a potential target for the treatment of both cancer anorexia and weight loss, as well as of obesity.
Subject(s)
Anorexia/metabolism , Cytokines/physiology , Multigene Family/immunology , Prostatic Neoplasms/metabolism , Weight Loss , Animals , Anorexia/genetics , Anorexia/immunology , Anorexia/physiopathology , Antibodies/administration & dosage , Antibodies/physiology , Cell Line, Tumor , Cytokines/blood , Cytokines/genetics , Cytokines/immunology , Growth Differentiation Factor 15 , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Prostatic Neoplasms/blood , Prostatic Neoplasms/immunology , Prostatic Neoplasms/physiopathology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunology , Transforming Growth Factor beta/physiology , Weight Loss/genetics , Weight Loss/immunologyABSTRACT
Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2-regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2-like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with "BH3 mimetic" drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.
Subject(s)
Apoptosis/physiology , Helminth Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Schistosoma japonicum/metabolism , Schistosoma mansoni/metabolism , Animals , Apoptosis/drug effects , Biphenyl Compounds/pharmacology , Crystallography, X-Ray , Helminth Proteins/antagonists & inhibitors , Helminth Proteins/genetics , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Nitrophenols/pharmacology , Peptide Fragments/pharmacology , Piperazines/pharmacology , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Schistosoma japonicum/genetics , Schistosoma mansoni/genetics , Schistosomiasis japonica/drug therapy , Schistosomiasis japonica/genetics , Schistosomiasis japonica/metabolism , Schistosomiasis mansoni/drug therapy , Schistosomiasis mansoni/genetics , Schistosomiasis mansoni/metabolism , Sulfonamides/pharmacologyABSTRACT
Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.
Subject(s)
Antineoplastic Agents , Apoptosis , Carbamates , Colorectal Neoplasms , Protein Kinase Inhibitors , bcl-X Protein , Animals , Humans , Mice , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , bcl-X Protein/antagonists & inhibitors , bcl-X Protein/metabolism , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Apoptosis/drug effectsABSTRACT
Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.